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1.  Nuclear Receptor Expression Defines a Set of Prognostic Biomarkers for Lung Cancer 
PLoS Medicine  2010;7(12):e1000378.
David Mangelsdorf and colleagues show that nuclear receptor expression is strongly associated with clinical outcomes of lung cancer patients, and this expression profile is a potential prognostic signature for lung cancer patient survival time, particularly for individuals with early stage disease.
Background
The identification of prognostic tumor biomarkers that also would have potential as therapeutic targets, particularly in patients with early stage disease, has been a long sought-after goal in the management and treatment of lung cancer. The nuclear receptor (NR) superfamily, which is composed of 48 transcription factors that govern complex physiologic and pathophysiologic processes, could represent a unique subset of these biomarkers. In fact, many members of this family are the targets of already identified selective receptor modulators, providing a direct link between individual tumor NR quantitation and selection of therapy. The goal of this study, which begins this overall strategy, was to investigate the association between mRNA expression of the NR superfamily and the clinical outcome for patients with lung cancer, and to test whether a tumor NR gene signature provided useful information (over available clinical data) for patients with lung cancer.
Methods and Findings
Using quantitative real-time PCR to study NR expression in 30 microdissected non-small-cell lung cancers (NSCLCs) and their pair-matched normal lung epithelium, we found great variability in NR expression among patients' tumor and non-involved lung epithelium, found a strong association between NR expression and clinical outcome, and identified an NR gene signature from both normal and tumor tissues that predicted patient survival time and disease recurrence. The NR signature derived from the initial 30 NSCLC samples was validated in two independent microarray datasets derived from 442 and 117 resected lung adenocarcinomas. The NR gene signature was also validated in 130 squamous cell carcinomas. The prognostic signature in tumors could be distilled to expression of two NRs, short heterodimer partner and progesterone receptor, as single gene predictors of NSCLC patient survival time, including for patients with stage I disease. Of equal interest, the studies of microdissected histologically normal epithelium and matched tumors identified expression in normal (but not tumor) epithelium of NGFIB3 and mineralocorticoid receptor as single gene predictors of good prognosis.
Conclusions
NR expression is strongly associated with clinical outcomes for patients with lung cancer, and this expression profile provides a unique prognostic signature for lung cancer patient survival time, particularly for those with early stage disease. This study highlights the potential use of NRs as a rational set of therapeutically tractable genes as theragnostic biomarkers, and specifically identifies short heterodimer partner and progesterone receptor in tumors, and NGFIB3 and MR in non-neoplastic lung epithelium, for future detailed translational study in lung cancer.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer, the most common cause of cancer-related death, kills 1.3 million people annually. Most lung cancers are “non-small-cell lung cancers” (NSCLCs), and most are caused by smoking. Exposure to chemicals in smoke causes changes in the genes of the cells lining the lungs that allow the cells to grow uncontrollably and to move around the body. How NSCLC is treated and responds to treatment depends on its “stage.” Stage I tumors, which are small and confined to the lung, are removed surgically, although chemotherapy is also sometimes given. Stage II tumors have spread to nearby lymph nodes and are treated with surgery and chemotherapy, as are some stage III tumors. However, because cancer cells in stage III tumors can be present throughout the chest, surgery is not always possible. For such cases, and for stage IV NSCLC, where the tumor has spread around the body, patients are treated with chemotherapy alone. About 70% of patients with stage I and II NSCLC but only 2% of patients with stage IV NSCLC survive for five years after diagnosis; more than 50% of patients have stage IV NSCLC at diagnosis.
Why Was This Study Done?
Patient responses to treatment vary considerably. Oncologists (doctors who treat cancer) would like to know which patients have a good prognosis (are likely to do well) to help them individualize their treatment. Consequently, the search is on for “prognostic tumor biomarkers,” molecules made by cancer cells that can be used to predict likely clinical outcomes. Such biomarkers, which may also be potential therapeutic targets, can be identified by analyzing the overall pattern of gene expression in a panel of tumors using a technique called microarray analysis and looking for associations between the expression of sets of genes and clinical outcomes. In this study, the researchers take a more directed approach to identifying prognostic biomarkers by investigating the association between the expression of the genes encoding nuclear receptors (NRs) and clinical outcome in patients with lung cancer. The NR superfamily contains 48 transcription factors (proteins that control the expression of other genes) that respond to several hormones and to diet-derived fats. NRs control many biological processes and are targets for several successful drugs, including some used to treat cancer.
What Did the Researchers Do and Find?
The researchers analyzed the expression of NR mRNAs using “quantitative real-time PCR” in 30 microdissected NSCLCs and in matched normal lung tissue samples (mRNA is the blueprint for protein production). They then used an approach called standard classification and regression tree analysis to build a prognostic model for NSCLC based on the expression data. This model predicted both survival time and disease recurrence among the patients from whom the tumors had been taken. The researchers validated their prognostic model in two large independent lung adenocarcinoma microarray datasets and in a squamous cell carcinoma dataset (adenocarcinomas and squamous cell carcinomas are two major NSCLC subtypes). Finally, they explored the roles of specific NRs in the prediction model. This analysis revealed that the ability of the NR signature in tumors to predict outcomes was mainly due to the expression of two NRs—the short heterodimer partner (SHP) and the progesterone receptor (PR). Expression of either gene could be used as a single gene predictor of the survival time of patients, including those with stage I disease. Similarly, the expression of either nerve growth factor induced gene B3 (NGFIB3) or mineralocorticoid receptor (MR) in normal tissue was a single gene predictor of a good prognosis.
What Do These Findings Mean?
These findings indicate that the expression of NR mRNA is strongly associated with clinical outcomes in patients with NSCLC. Furthermore, they identify a prognostic NR expression signature that provides information on the survival time of patients, including those with early stage disease. The signature needs to be confirmed in more patients before it can be used clinically, and researchers would like to establish whether changes in mRNA expression are reflected in changes in protein expression if NRs are to be targeted therapeutically. Nevertheless, these findings highlight the potential use of NRs as prognostic tumor biomarkers. Furthermore, they identify SHP and PR in tumors and two NRs in normal lung tissue as molecules that might provide new targets for the treatment of lung cancer and new insights into the early diagnosis, pathogenesis, and chemoprevention of lung cancer.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000378.
The Nuclear Receptor Signaling Atlas (NURSA) is consortium of scientists sponsored by the US National Institutes of Health that provides scientific reagents, datasets, and educational material on nuclear receptors and their co-regulators to the scientific community through a Web-based portal
The Cancer Prevention and Research Institute of Texas (CPRIT) provides information and resources to anyone interested in the prevention and treatment of lung and other cancers
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small-cell carcinoma and on tumor markers (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
Wikipedia has a page on nuclear receptors (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1000378
PMCID: PMC3001894  PMID: 21179495
2.  Aromatase Expression Predicts Survival in Women with Early-Stage Non–Small Cell Lung Cancer 
Cancer research  2007;67(21):10484-10490.
Estrogen signaling is critical in the progression of tumors that bear estrogen receptors. In most patients with breast cancer, inhibitors that block interactions of estrogen with its receptors or suppress the production of endogenous estrogens are important interventions in the clinic. Recent evidence now suggests that estrogen also contributes to the pathogenesis of non–small cell lung cancer (NSCLC). We used a human lung cancer xenograph model system to analyze the effect of aromatase or estradiol on tumor growth. We further examined the level of protein expression of aromatase in 422 patients with NSCLC using a high-density tissue microarray. Results were confirmed and validated on an independent patient cohort (n = 337). Lower levels of aromatase predicted a greater chance of survival in women 65 years and older. Within this population, the prognostic value of aromatase was greatest in earlier stage lung cancer (stage I/II). In addition, for women with no history of smoking, lower aromatase levels were a strong predictor of survival. Our findings implicate aromatase as an early-stage predictor of survival in some women with NSCLC. We predict that women whose lung cancers have higher levels of aromatase might be good candidates for targeted treatment with aromatase inhibitors.
doi:10.1158/0008-5472.CAN-07-2607
PMCID: PMC3581354  PMID: 17974992
3.  The role of estrogen, progesterone and aromatase in human non-small-cell lung cancer 
Lung cancer management  2012;1(4):259-272.
SUMMARY
Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide. Despite advances in treatment, patients have few effective therapeutic options and survival rates remain low. Emerging evidence suggests that the hormones estrogen and progesterone play a key role in the progression of non-small-cell lung cancer (NSCLC). The aromatase enzyme, which is responsible for a key step in estrogen biosynthesis, elicits higher levels of estrogen in lung tumors as well as in metastases compared with nonmalignant tissues. Thus, aromatase may prove to be a key predictive biomarker for treatment of NSCLC. Epidemiologic and preclinical data show estrogens play a critical role in lung tumor development and progression. Two estrogen receptors, α and β, are expressed in normal and in cancerous lung epithelium, and estrogen promotes gene transcription that stimulates cell proliferation and inhibits cell death. Furthermore, expression of both forms of estrogen receptor, progesterone receptor and aromatase in NSCLC specimens has been correlated with worse clinical outcomes. Combination therapies that include estrogen receptor downregulators and aromatase inhibitors are currently being assessed in Phase I–II clinical trials among patients with advanced NSCLC. Results will help guide future lung cancer management decisions, with a goal of achieving more effective and less toxic treatments for patients.
doi:10.2217/lmt.12.44
PMCID: PMC3643508  PMID: 23650476
4.  Expression Levels of Estrogen Receptor Beta in Conjunction with Aromatase Predict Survival in Non-Small Cell Lung Cancer 
Estrogen signaling pathways may play a significant role in the pathogenesis of non-small cell lung cancers (NSCLC) as evidenced by the expression of aromatase and estrogen receptors (ERα and ERβ) in many of these tumors. Here we examine whether ERα and ERβ levels in conjunction with aromatase define patient groups with respect to survival outcomes and possible treatment regimens. Immunohistochemistry was performed on a high-density tissue microarray with resulting data and clinical information available for 377 patients. Patients were subdivided by gender, age and tumor histology, and survival data was determined using the Cox proportional hazards model and Kaplan-Meier curves. Neither ERα nor ERβ alone were predictors of survival in NSCLC. However, when coupled with aromatase expression, higher ERβ levels predicted worse survival in patients whose tumors expressed higher levels of aromatase. Although this finding was present in patients of both genders, it was especially pronounced in women ≥ 65 years old, where higher expression of both ERβ and aromatase indicated a markedly worse survival rate than that determined by aromatase alone. Conclusion: Expression of ERβ together with aromatase has predictive value for survival in different gender and age subgroups of NSCLC patients. This predictive value is stronger than each individual marker alone. Our results suggest treatment with aromatase inhibitors alone or combined with estrogen receptor modulators may be of benefit in some subpopulations of these patients.
doi:10.1016/j.lungcan.2011.03.009
PMCID: PMC3175023  PMID: 21511357
NSCLC; tissue microarray; aromatase; estrogen receptor; immunohistochemistry; prognosis
5.  Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small cell lung cancer 
Introduction
Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC.
Methods
We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway either individually or in combination.
Results
The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor (PR)-A, PR-B and aromatase proteins are expressed in all lines to varying degrees, with trends towards lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited.
Conclusions
These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition.
doi:10.1097/JTO.0b013e31827d525c
PMCID: PMC3573351  PMID: 23399957
epidermal growth factor receptor; estrogen; estrogen receptor; lung cancer; fulvestrant
6.  Targeting Aromatase and Estrogen Signaling in Human Non-Small Cell Lung Cancer 
Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non-small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long-term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor-associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.
doi:10.1111/j.1749-6632.2009.04116.x
PMCID: PMC2782616  PMID: 19250205
non-small cell lung cancer (NSCLC); aromatase; CYP19; estrogen receptor (ER); epidermal growth factor receptor (EGFR); vascular endothelial growth factor (VEGF) receptor; anastrazole; exemestane; fulvestrant; vandetanib
7.  Intratumoral localization and activity of 17β-hydroxysteroid dehydrogenase type 1 in non-small cell lung cancer: a potent prognostic factor 
Background
Estrogens were recently demonstrated to be synthesized in non-small cell lung carcinomas (NSCLCs) via aromatase activity and aromatase inhibitor (AI) did suppressed estrogen receptor (ER) positive NSCLC growth. However, other enzymes involved in intratumoral production and metabolism of estrogens, i.e. 17β-hydroxysteroid dehydrogenases (i.e. 17βHSD1 and 17βHSD2) and others have not been studied. Therefore, in this study, we examined the clinical/ biological significance of 17β-hydroxysteroid dehydrogenases in NSCLCs.
Methodology
Archival materials obtained from 103 NSCLC patients were immunohistochemically evaluated using anti-17βHSD1 and anti-17βHSD2 antibodies. The findings of immunohistochemistry were then correlated with intratumoral estrone (E1) and estradiol (E2) concentration, clinicopathological factors and overall survival of the patients. We further employed NSCLC cell lines, A549 and LK87 to study the functional significance of 17βHSD1, in vitro.
Results
A higher 17βHSD1 immunoreactivity tended to be positively associated with aromatase (p=0.057) and tumor stage (p=0.055) whereas a higher 17βHSD2 immunoreactivity was positively associated with a squamous cell and adenosquamous cell carcinomas subtypes (p=0.031), tumor stage (p=0.004), T factor of TNM classification (p=0.010), maximum tumor diameter (p=0.002) and tended to be associated with N factor of TMN classification (p=0.065). A higher 17βHSD1 immunoreactivity was also significantly associated with lower intratumoral E1 concentration (p=0.040) and a higher intratumoral E2/E1 concentration ratio (p=0.028). On the other hand a higher 17βHSD2 immunoreactivity was significantly associated with higher intratumoral E1 concentration (p=0.035). Results of multivariate regression analysis demonstrated an increased 17βHSD1 immunoreactivity in tumor cells as an independent negative prognostic factor (HR= 2.83, p=0.007). E1 treatment in 17βHSD1 positive NSCLC cells, A549 and LK87, resulted in E2 production (p<0.0001) and enhanced cell proliferation, which was abrogated effectively by 17βHSD1 siRNA knockdown (p<0.0001). In addition, aromatase inhibitor treatment resulted in 17βHSD1 up regulation in both A549 and LK87 cells.
Conclusion
Results of our present study suggest that 17βHSD1 may be considered an important prognostic factor in NSCLC patients and targeting 17βHSD1 activity may further improve the clinical response in estrogen responsive NSCLC patients.
doi:10.1186/1479-5876-11-167
PMCID: PMC3724709  PMID: 23837683
Lung cancer; Intratumoral estrogens; 17β-hydroxysteroid dehydrogenase; Targeted therapy
8.  A Gene Expression Signature Predicts Survival of Patients with Stage I Non-Small Cell Lung Cancer 
PLoS Medicine  2006;3(12):e467.
Background
Lung cancer is the leading cause of cancer-related death in the United States. Nearly 50% of patients with stages I and II non-small cell lung cancer (NSCLC) will die from recurrent disease despite surgical resection. No reliable clinical or molecular predictors are currently available for identifying those at high risk for developing recurrent disease. As a consequence, it is not possible to select those high-risk patients for more aggressive therapies and assign less aggressive treatments to patients at low risk for recurrence.
Methods and Findings
In this study, we applied a meta-analysis of datasets from seven different microarray studies on NSCLC for differentially expressed genes related to survival time (under 2 y and over 5 y). A consensus set of 4,905 genes from these studies was selected, and systematic bias adjustment in the datasets was performed by distance-weighted discrimination (DWD). We identified a gene expression signature consisting of 64 genes that is highly predictive of which stage I lung cancer patients may benefit from more aggressive therapy. Kaplan-Meier analysis of the overall survival of stage I NSCLC patients with the 64-gene expression signature demonstrated that the high- and low-risk groups are significantly different in their overall survival. Of the 64 genes, 11 are related to cancer metastasis (APC, CDH8, IL8RB, LY6D, PCDHGA12, DSP, NID, ENPP2, CCR2, CASP8, and CASP10) and eight are involved in apoptosis (CASP8, CASP10, PIK3R1, BCL2, SON, INHA, PSEN1, and BIK).
Conclusions
Our results indicate that gene expression signatures from several datasets can be reconciled. The resulting signature is useful in predicting survival of stage I NSCLC and might be useful in informing treatment decisions.
Meta-analysis of several lung cancer gene expression studies yields a set of 64 genes whose expression profile is useful in predicting survival of patients with early-stage lung cancer and possibly informing treatment decisions.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC) and are mainly caused by smoking. Like other cancers, how NSCLC is treated depends on the “stage” at which it is detected. Stage IA NSCLCs are small and confined to the lung and can be removed surgically; patients with slightly larger stage IB tumors often receive chemotherapy after surgery. In stage II NSCLC, cancer cells may be present in lymph nodes near the tumor. Surgery plus chemotherapy is the usual treatment for this stage and for some stage III NSCLCs. However, in this stage, the tumor can be present throughout the chest and surgery is not always possible. For such cases and in stage IV NSCLC, where the tumor has spread throughout the body, patients are treated with chemotherapy alone. The stage at which NSCLC is detected also determines how well patients respond to treatment. Those who can be treated surgically do much better than those who can't. So, whereas only 2% of patients with stage IV lung cancer survive for 5 years after diagnosis, about 70% of patients with stage I or II lung cancer live at least this long.
Why Was This Study Done?
Even stage I and II lung cancers often recur and there is no accurate way to identify the patients in which this will happen. If there was, these patients could be given aggressive chemotherapy, so the search is on for a “molecular signature” to help identify which NSCLCs are likely to recur. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral differences are caused by changes in their genetic material that alter their patterns of RNA transcription and protein expression. In this study, the researchers have investigated whether data from several microarray studies (a technique used to catalog the genes expressed in cells) can be pooled to construct a gene expression signature that predicts the survival of patients with stage I NSCLC.
What Did the Researchers Do and Find?
The researchers took the data from seven independent microarray studies (including a new study of their own) that recorded gene expression profiles related to survival time (less than 2 years and greater than 5 years) for stage I NSCLC. Because these studies had been done in different places with slightly different techniques, the researchers applied a statistical tool called distance-weighted discrimination to smooth out any systematic differences among the studies before identifying 64 genes whose expression was associated with survival. Most of these genes are involved in cell adhesion, cell motility, cell proliferation, and cell death, all processes that are altered in cancer cells. The researchers then developed a statistical model that allowed them to use the gene expression and survival data to calculate risk scores for nearly 200 patients in five of the datasets. When they separated the patients into high and low risk groups on the basis of these scores, the two groups were significantly different in terms of survival time. Indeed, the gene expression signature was better at predicting outcome than routine staging. Finally, the researchers validated the gene expression signature by showing that it predicted survival with more than 85% accuracy in two independent datasets.
What Do These Findings Mean?
The 64 gene expression signature identified here could help clinicians prepare treatment plans for patients with stage I NSCLC. Because it accurately predicts survival in patients with adenocarcinoma or squamous cell cancer (the two major subtypes of NSCLC), it potentially indicates which of these patients should receive aggressive chemotherapy and which can be spared this unpleasant treatment. Previous attempts to establish gene expression signatures to predict outcome have used data from small groups of patients and have failed when tested in additional patients. In contrast, this new signature seems to be generalizable. Nevertheless, its ability to predict outcomes must be confirmed in further studies before it is routinely adopted by oncologists for treatment planning.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030467.
US National Cancer Institute information on lung cancer for patients and health professionals.
MedlinePlus encyclopedia entries on small-cell and non-small-cell lung cancer.
Cancer Research UK, information on patients about all aspects of lung cancer.
Wikipedia pages on DNA microarrays and expression profiling (note that Wikipedia is a free online encyclopedia that anyone can edit).
doi:10.1371/journal.pmed.0030467
PMCID: PMC1716187  PMID: 17194181
9.  Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma 
PLoS Medicine  2010;7(7):e1000315.
William Lockwood and colleagues show that the focal amplification of a gene, BRF2, on Chromosome 8p12 plays a key role in squamous cell carcinoma of the lung.
Background
Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes—adenocarcinoma (AC) and squamous cell carcinoma (SqCC)—respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome.
Methods and Findings
We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage.
Conclusions
This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer is the commonest cause of cancer-related death. Every year, 1.3 million people die from this disease, which is mainly caused by smoking. Most cases of lung cancer are “non-small cell lung cancers” (NSCLCs). Like all cancers, NSCLC starts when cells begin to divide uncontrollably and to move round the body (metastasize) because of changes (mutations) in their genes. These mutations are often in “oncogenes,” genes that, when activated, encourage cell division. Oncogenes can be activated by mutations that alter the properties of the proteins they encode or by mutations that increase the amount of protein made from them, such as gene amplification (an increase in the number of copies of a gene). If NSCLC is diagnosed before it has spread from the lungs (stage I disease), it can be surgically removed and many patients with stage I NSCLC survive for more than 5 years after their diagnosis. Unfortunately, in more than half of patients, NSCLC has metastasized before it is diagnosed. This stage IV NSCLC can be treated with chemotherapy (toxic chemicals that kill fast-growing cancer cells) but only 2% of patients with stage IV lung cancer are alive 5 years after diagnosis.
Why Was This Study Done?
Traditionally, NSCLC has been regarded as a single disease in terms of treatment. However, emerging evidence suggests that the two major subtypes of NSCLC—adenocarcinoma and squamous cell carcinoma (SqCC)—respond differently to chemotherapy. Adenocarcinoma and SqCC start in different types of lung cell and experts think that for each cell type in the body, specific combinations of mutations interact with the cell type's own unique characteristics to provide the growth and survival advantage needed for cancer development. If this is true, then identifying the molecular differences between adenocarcinoma and SqCC could provide targets for more effective therapies for these major subtypes of NSCLC. Amplification of a chromosome region called 8p12 is very common in NSCLC, which suggests that an oncogene that drives lung cancer development is present in this chromosome region. In this study, the researchers investigate this possibility by looking for an amplified gene in the 8p12 chromosome region that makes increased amounts of protein in lung SqCC but not in lung adenocarcinoma.
What Did the Researchers Do and Find?
The researchers used a technique called comparative genomic hybridization to show that focal regions of Chromosome 8p are amplified in about 40% of lung SqCCs, but that DNA loss in this region is the most common alteration in lung adenocarcinomas. Ten genes in the 8p12 chromosome region were expressed at higher levels in the SqCC samples that they examined than in adenocarcinoma samples, they report, and overexpression of five of these genes correlated with amplification of the 8p12 region in the SqCC samples. Only one of the genes—BRF2—was more highly expressed in squamous carcinoma cells than in normal bronchial epithelial cells (the cell type that lines the tubes that take air into the lungs and from which SqCC develops). Artificially induced expression of BRF2 in bronchial epithelial cells made these normal cells behave like tumor cells, whereas reduction of BRF2 expression in squamous carcinoma cells made them behave more like normal bronchial epithelial cells. Finally, BRF2 was frequently activated in two early stages of squamous cell carcinoma—bronchial carcinoma in situ and dysplastic lesions.
What Do These Findings Mean?
Together, these findings show that the focal amplification of chromosome region 8p12 plays a role in the development of lung SqCC but not in the development of lung adenocarcinoma, the other major subtype of NSCLC. These findings identify BRF2 (which encodes a RNA polymerase III transcription initiation factor, a protein that is required for the synthesis of RNA molecules that help to control cell growth) as a lung SqCC-specific oncogene and uncover a unique mechanism for lung SqCC development. Most importantly, these findings suggest that genetic activation of BRF2 could be used as a marker for lung SqCC, which might facilitate the early detection of this type of NSCLC and that BRF2 might provide a new target for therapy.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000315.
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small cell carcinoma (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
doi:10.1371/journal.pmed.1000315
PMCID: PMC2910599  PMID: 20668658
10.  Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer 
BMC Cancer  2012;12:624.
Background
G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer.
Methods
The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed.
Results
Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue.
Conclusion
The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.
doi:10.1186/1471-2407-12-624
PMCID: PMC3557142  PMID: 23273253
GPER; GPR30; Estrogen; Estrogen receptor; Lung cancer; Protein expression; Immunohistochemistry; Tissue microarray
11.  Small-Animal PET of Steroid Hormone Receptors Predicts Tumor Response to Endocrine Therapy Using a Preclinical Model of Breast Cancer 
Estrogen receptor-α (ERα) and progesterone receptor (PR) are expressed in most human breast cancers and are important predictive factors for directing therapy. Because of de novo and acquired resistance to endocrine therapy, there remains a need to identify which ERα-positive (ERα+)/PR-positive (PR+) tumors are most likely to respond. The purpose of this study was to use estrogen- and progestin-based radiopharmaceuticals to image ERα and PR in mouse mammary tumors at baseline and after hormonal therapy and to determine whether changes in these imaging biomarkers can serve as an early predictive indicator of therapeutic response.
Methods
Mammary adenocarcinomas that spontaneously develop in aged female mice deficient in signal transducer and activator of transcription-1 (STAT1) were used. Imaging of ERα and PR in primary tumor–bearing mice and mice implanted with mammary cell lines (SSM1, SSM2, and SSM3) derived from primary STAT1-deficient (STAT1−/−) tumors was performed. Hormonal treatments consisted of estradiol, an ER agonist; letrozole, an aromatase inhibitor; and fulvestrant, a pure ER antagonist. Small-animal PET/CT was performed using 18F-fluoroestradiol (18F-FES) for ER, 18F-fluoro furanyl norprogesterone (18F-FFNP) for PR, and 18F-FDG for glucose uptake. Tracer uptake in the tumor was quantified and compared with receptor concentration determined by in vitro assays of resected tumors.
Results
Primary STAT1−/− mammary tumors and implanted SSM2 and SSM3 tumors showed high 18F-FES and 18F-FFNP uptake and were confirmed to be ERα+/PR+. Classic estrogen-induced regulation of the progesterone receptor gene was demonstrated by increased 18F-FFNP uptake of estradiol-treated SSM3 tumors. Treatment with fulvestrant decreased 18F-FFNP, 18F-FES, and 18F-FDG uptake and inhibited growth of SSM3 tumors but decreased only 18F-FES uptake in SSM2 tumors, with no effect on growth, despite both tumors being ERα+/PR+. Decreased 18F-FFNP uptake by SSM3 tumors occurred early after initiation of treatment, before measurable tumor growth inhibition.
Conclusion
Using small-animal PET, a profile was identified that distinguished fulvestrant-sensitive from fulvestrant-resistant ERα+/PR+ tumors before changes in tumor size. This work demonstrates that imaging baseline tumoral 18F-FES uptake and initial changes in 18F-FFNP uptake in a non-invasive manner is a potentially useful strategy to identify responders and nonresponders to endocrine therapy at an early stage.
doi:10.2967/jnumed.112.103465
PMCID: PMC3956595  PMID: 22669982
PET imaging; estrogen receptor; progesterone receptor; breast cancer; endocrine therapy
12.  Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation 
Purpose
To determine the frequency of estrogen receptor α and β and progesterone receptor protein immunohistochemical expression in a large set of non–small cell lungcarcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports.
Experimental Design
Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors α and β and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients’ clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status.
Results
Estrogen receptors α and β were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for α and 42-56% for β; cytoplasm: <1-42% for α and 20-98% for β) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor α nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor α expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor α expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor β and progesterone receptor but not estrogen receptor α expressed in the normal epithelium adjacent to lung adenocarcinomas.
Conclusions
Estrogen receptor α and β expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor α expression correlates with EGFR mutations.
doi:10.1158/1078-0432.CCR-09-0033
PMCID: PMC2893045  PMID: 19706809
13.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
14.  Aberrant DNA Methylation of OLIG1, a Novel Prognostic Factor in Non-Small Cell Lung Cancer 
PLoS Medicine  2007;4(3):e108.
Background
Lung cancer is the leading cause of cancer-related death worldwide. Currently, tumor, node, metastasis (TNM) staging provides the most accurate prognostic parameter for patients with non-small cell lung cancer (NSCLC). However, the overall survival of patients with resectable tumors varies significantly, indicating the need for additional prognostic factors to better predict the outcome of the disease, particularly within a given TNM subset.
Methods and Findings
In this study, we investigated whether adenocarcinomas and squamous cell carcinomas could be differentiated based on their global aberrant DNA methylation patterns. We performed restriction landmark genomic scanning on 40 patient samples and identified 47 DNA methylation targets that together could distinguish the two lung cancer subgroups. The protein expression of one of those targets, oligodendrocyte transcription factor 1 (OLIG1), significantly correlated with survival in NSCLC patients, as shown by univariate and multivariate analyses. Furthermore, the hazard ratio for patients negative for OLIG1 protein was significantly higher than the one for those patients expressing the protein, even at low levels.
Conclusions
Multivariate analyses of our data confirmed that OLIG1 protein expression significantly correlates with overall survival in NSCLC patients, with a relative risk of 0.84 (95% confidence interval 0.77–0.91, p < 0.001) along with T and N stages, as indicated by a Cox proportional hazard model. Taken together, our results suggests that OLIG1 protein expression could be utilized as a novel prognostic factor, which could aid in deciding which NSCLC patients might benefit from more aggressive therapy. This is potentially of great significance, as the addition of postoperative adjuvant chemotherapy in T2N0 NSCLC patients is still controversial.
Christopher Plass and colleagues find thatOLIG1 expression correlates with survival in lung cancer patients and suggest that it could be used in deciding which patients are likely to benefit from more aggressive therapy.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC). Like other cancers, treatment of NCSLC depends on the “TNM stage” at which the cancer is detected. Staging takes into account the size and local spread of the tumor (its T classification), whether nearby lymph nodes contain tumor cells (its N classification), and whether tumor cells have spread (metastasized) throughout the body (its M classification). Stage I tumors are confined to the lung and are removed surgically. Stage II tumors have spread to nearby lymph nodes and are treated with a combination of surgery and chemotherapy. Stage III tumors have spread throughout the chest, and stage IV tumors have metastasized around the body; patients with both of these stages are treated with chemotherapy alone. About 70% of patients with stage I or II lung cancer, but only 2% of patients with stage IV lung cancer, survive for five years after diagnosis.
Why Was This Study Done?
TNM staging is the best way to predict the likely outcome (prognosis) for patients with NSCLC, but survival times for patients with stage I and II tumors vary widely. Another prognostic marker—maybe a “molecular signature”—that could distinguish patients who are likely to respond to treatment from those whose cancer will inevitably progress would be very useful. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral changes are caused by alterations in the pattern of proteins expressed by the cells. But what causes these alterations? The answer in some cases is “epigenetic changes” or chemical modifications of genes. In cancer cells, methyl groups are aberrantly added to GC-rich gene regions. These so-called “CpG islands” lie near gene promoters (sequences that control the transcription of DNA into mRNA, the template for protein production), and their methylation stops the promoters working and silences the gene. In this study, the researchers have investigated whether aberrant methylation patterns vary between NSCLC subtypes and whether specific aberrant methylations are associated with survival and can, therefore, be used prognostically.
What Did the Researchers Do and Find?
The researchers used “restriction landmark genomic scanning” (RLGS) to catalog global aberrant DNA methylation patterns in human lung tumor samples. In RLGS, DNA is cut into fragments with a restriction enzyme (a protein that cuts at specific DNA sequences), end-labeled, and separated using two-dimensional gel electrophoresis to give a pattern of spots. Because methylation stops some restriction enzymes cutting their target sequence, normal lung tissue and lung tumor samples yield different patterns of spots. The researchers used these patterns to identify 47 DNA methylation targets (many in CpG islands) that together distinguished between adenocarcinomas and squamous cell carcinomas, two major types of NSCLCs. Next, they measured mRNA production from the genes with the greatest difference in methylation between adenocarcinomas and squamous cell carcinomas. OLIG1 (the gene that encodes a protein involved in nerve cell development) had one of the highest differences in mRNA production between these tumor types. Furthermore, three-quarters of NSCLCs had reduced or no expression of OLIG1 protein and, when the researchers analyzed the association between OLIG1 protein expression and overall survival in patients with NSCLC, reduced OLIG1 protein expression was associated with reduced survival.
What Do These Findings Mean?
These findings indicate that different types of NSCLC can be distinguished by examining their aberrant methylation patterns. This suggests that the establishment of different DNA methylation patterns might be related to the cell type from which the tumors developed. Alternatively, the different aberrant methylation patterns might reflect the different routes that these cells take to becoming tumor cells. This research identifies a potential new prognostic marker for NSCLC by showing that OLIG1 protein expression correlates with overall survival in patients with NSCLC. This correlation needs to be tested in a clinical setting to see if adding OLIG1 expression to the current prognostic parameters can lead to better treatment choices for early-stage lung cancer patients and ultimately improve these patients' overall survival.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040108.
Patient and professional information on lung cancer, including staging (in English and Spanish), is available from the US National Cancer Institute
The MedlinePlus encyclopedia has pages on non-small cell lung cancer (in English and Spanish)
Cancerbackup provides patient information on lung cancer
CancerQuest, provided by Emory University, has information about how cancer develops (in English, Spanish, Chinese and Russian)
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence gives background information and the latest news about epigenetics (in several European languages)
doi:10.1371/journal.pmed.0040108
PMCID: PMC1831740  PMID: 17388669
15.  Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics 
PLoS Medicine  2007;4(10):e316.
Background
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.
Methods and Findings
We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.
Conclusions
Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation.
Editors' Summary
Background.
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC.
Why Was This Study Done?
Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR.
What Did the Researchers Do and Find?
The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040316.
A perspective by Ingo Mellinghoff discusses this article and two related research articles
Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
CancerQuest provides information on all aspects of cancer from Emory University (in several languages)
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040316
PMCID: PMC2043013  PMID: 17973573
16.  Changes in Aromatase (CYP19) Gene Promoter Usage in Non-Small Cell Lung Cancer 
In humans, aromatase (CYP19) gene expression is regulated via alternative promoters. Activation of each promoter gives rise to a CYP19 mRNA species with a unique 5′-untranslated region. Inhibition of aromatase has been reported to downregulate lung tumor growth. The genetic basis for CYP19 gene expression and aromatase activity in lung cancer remains poorly understood. We analyzed tissues from 15 patients with non-small cell lung cancer (NSCLC) to evaluate CYP19 promoter usage and promoter-specific aromatase mRNA levels in NSCLC tumor tissues and adjacent non-malignant tissues. CYP19 promoter usage was determined by multiplex RT-PCR and aromatase mRNA levels were measured with real-time RT-PCR. In non-malignant tissues, aromatase mRNA was primarily derived from activation of CYP19 promoter I.4. Although promoter I.4 usage was also dominant in tumor tissues, I.4 activation was significantly lower compared with adjacent non-malignant tissues. Activity of promoters I.3, I.1 and I.7 was significantly higher in tumor tissues compared with non-malignant tissues. In 4 of 15 cases of non-small cell lung cancer, switching from CYP19 promoter I.4 to the alternative promoters II, I.1 or I.7 was observed. In 9 cases, there were significantly higher levels of aromatase mRNA in lung tumor tissues compared with adjacent non-malignant tissues. These findings suggest aberrant activation of alternative CYP19 promoters that may lead to upregulation of local aromatase expression in some cases of NSCLC. Further studies are needed to examine the impact of alternative CYP19 promoter usage on local estrogen levels and lung tumor growth.
doi:10.1016/j.lungcan.2011.01.003
PMCID: PMC3118270  PMID: 21320735
non-small cell lung cancer; estrogen; aromatase; CYP19; promoter; promoter usage
17.  Combining the Multi-Targeted Tyrosine Kinase Inhibitor Vandetanib with the Anti-Estrogen Fulvestrant Enhances its Anti-tumor Effect in Non-Small Cell Lung Cancer 
Journal of Thoracic Oncology  2012;7(3):485-495.
Introduction
Estrogen is known to promote proliferation and to activate the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). Vascular endothelial growth factor (VEGF) is a known estrogen responsive gene in breast cancer. We sought to determine if the VEGF pathway is also regulated by estrogen in lung cancer cells, and whether combining an inhibitor of the ER pathway with a dual VEGFR/EGFR inhibitor would show enhanced anti-tumor effects.
Methods
We examined activation of EGFR and expression of VEGF in response to β-estradiol, and the anti-tumor activity of the multi-targeted VEGFR/EGFR/RET inhibitor, vandetanib, when combined with the anti-estrogen fulvestrant both in vitro and in vivo.
Results
NSCLC cells expressed VEGFR-3 and EGFR. Vandetanib treatment of NSCLC cells resulted in inhibition of EGFR and VEGFR-3 and inhibition of β-estradiol-induced P-MAPK activation, demonstrating that vandetanib blocks β-estradiol-induced EGFR signaling. Treatment with β-estradiol stimulated VEGFA mRNA and protein (P<0.0001 over baseline), suggesting estrogenic signaling causes heightened VEGFA pathway activation. This estrogenic induction of VEGFA mRNA appears largely dependent on cross-talk with EGFR. Long-term vandetanib treatment also significantly increased ERβ protein expression. The combination of vandetanib with fulvestrant maximally inhibited cell growth compared to single agents (P<0.0001) and decreased tumor xenograft volume by 64%, compared to 51% for vandetanib (P<0.05) and 23% for fulvestrant (P<0.005). Antitumor effects of combination therapy were accompanied by a significant increase in apoptotic cells compared to single agents.
Conclusions
Fulvestrant may enhance effects of vandetanib in NSCLC by blocking estrogen-driven activation of the EGFR pathway.
doi:10.1097/JTO.0b013e31824177ea
PMCID: PMC3288546  PMID: 22258476
EGFR; Estrogen; NSCLC; VEGF; VEGFR; Tyrosine kinase inhibitor
18.  BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations  
PLoS Medicine  2007;4(10):e315.
Background
Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.
Methods and Findings
To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.
Conclusions
Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.
Susumu Kobayashi and colleagues provide evidence that the polypeptide BIM is involved in tyrosine kinase inhibitor (TKI)-induced apoptosis in sensitiveEGFR-mutant cells and suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC). Many patients with NSCLC die within a year of their diagnosis, but recently, “targeted” therapies have increased the life expectancy of some of them. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genes. Targeted therapies specifically attack these changes and, unlike standard chemotherapy drugs, kill cancer cells without damaging normal cells. The targeted drugs used to treat NSCLC are gefitinib and erlotinib, two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In normal cells, messenger proteins bind to EGFR and activate its tyrosine kinase, an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These “phosphorylated” proteins then tell the cell to divide. In some NSCLCs, EGFR drives uncontrolled cell division because its tyrosine kinase is mutated and the cancer becomes dependent on or “addicted” to EGFR signaling for its survival. TKI treatment can dramatically shrink this subset of NSCLCs, most of which lack a specific part of EGFR (the gene that encodes EGFR) or have the amino acid leucine instead of arginine at position 858 (an L858R mutation) of EGFR.
Why Was This Study Done?
TKI-sensitive NSCLCs eventually become resistant to TKIs because they acquire additional (secondary) mutations. In half of these TKI-resistant tumors, the additional mutation is replacement of threonine by methionine at position 790 (T790M) in EGFR. However, the mutations responsible for the remaining cases of TKI resistance are not known. In addition, little is known about how TKIs induce cell death other than that they induce a type of cell death called apoptosis. A better understanding of how TKIs kill tumor cells and how secondary mutations block their effects could reveal ways to enhance their action and improve the outcome for patients with NSCLC. In this study, the researchers have studied the mechanism of TKI-induced cell death and of resistance to TKIs.
What Did the Researchers Do and Find?
The researchers first measured the ability of gefitinib to cause apoptosis (genetically programmed cell death) in NSCLC cell lines (tumor cells adapted to grow indefinitely in dishes) that had the EGFR deletion, the L858R mutation, or normal EGFR. Gefitinib caused apoptosis only in cell lines with altered EGFR. Then they asked whether a proapoptotic protein called BIM (a member of the BCL2 family of pro- and antiapoptotic proteins) is involved in TKI-induced cell death—BIM is known to be involved in this process in leukemia (blood cancer) cells. Gefitinib treatment increased the expression of BIM in TKI-sensitive NSCLC cell lines and reduced the phosphorylation of BIM (which makes BIM more active). By contrast, blocking BIM expression using a technique called RNA interference reduced TKI-induced apoptosis in TKI-sensitive NSCLC cells. Furthermore, introduction of the T790M resistance mutation into these cells blocked gefitinib-induced up-regulation of BIM and apoptosis. Finally, the researchers identified a new TKI resistance mutation (L747S, substitution of serine for leucine at position 747) in a patient whose TKI-sensitive NSCLC had become resistant to gefitinib, and showed that this resistance mutation also reduced TKI-induced apoptosis in cells growing in dishes by interfering with BIM up-regulation.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Cragg et al.) show that BIM is required for TKI-induced apoptosis in EGFR mutant NSCLC cells. They also show that mutations that make TKI-sensitive cells resistant to these drugs reduce TKI-induced apoptosis by preventing the upregulation of BIM. These results were obtained by examining the behavior of established cell lines growing in dishes and need to be confirmed in cells freshly isolated from tumors and in tumors themselves. However, they suggest that the efficacy of TKIs could be increased by finding ways to increase BIM expression or to activate other proteins involved in apoptosis Such approaches might be particularly beneficial for patients with NSCLC whose initially TKI-sensitive tumors have acquired mutations that make them resistant to TKIs.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040315.
Ingo Mellinghoff discusses this paper and two related ones in a perspective article
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer, including information on treatment with TKIs
CancerQuest information on all aspects of cancer from Emory University (in several languages)
Wikipedia pages on apoptosis, epidermal growth factor receptor, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040315
PMCID: PMC2043012  PMID: 17973572
19.  Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses 
Proteome Science  2011;9:60.
Background
A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E2) are distinct from those in non-E2-responsive cells.
Methods
FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E2 proliferative H1793 and non-E2-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.
Results
LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E2 or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.
Conclusion
Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.
doi:10.1186/1477-5956-9-60
PMCID: PMC3192725  PMID: 21951318
20.  Antiproliferative effect of exemestane in lung cancer cells 
Molecular Cancer  2009;8:109.
Background
Recent evidence suggests that estrogen signaling may be involved in the pathogenesis of non-small cell lung cancer (NSCLC). Aromatase is an enzyme complex that catalyses the final step in estrogen synthesis and is present in several tissues, including the lung. In the current study we investigated the activity of the aromatase inhibitor exemestane in human NSCLC cell lines H23 and A549.
Results
Aromatase expression was detected in both cell lines. H23 cells showed lower protein and mRNA levels of aromatase, compared to A549 cells. Exemestane decreased cell proliferation and increased apoptosis in both cell lines, 48 h after its application, with A549 exhibiting higher sensitivity than H23 cells. Aromatase protein and mRNA levels were not affected by exemestane in A549 cells, whereas an increase in both protein and mRNA levels was observed in H23 cells, 48 h after exemestane application. Moreover, an increase in cAMP levels was found in both cell lines, 15 min after the administration of exemestane. In addition, we studied the effect of exemestane on epidermal growth factor receptor (EGFR) localization and activation. Exemestane increased EGFR activation 15 min after its application in H23 cells. Furthermore, we demonstrated a translocation of EGFR from cell membrane, 24 h after the addition of exemestane in H23 cells. No changes in EGFR activation or localization were observed in A549 cells.
Conclusion
Our findings suggest an antiproliferative effect of exemestane on NSCLC cell lines. Exemestane may be more effective in cells with higher aromatase levels. Further studies are needed to assess the activity of exemestane in NSCLC.
doi:10.1186/1476-4598-8-109
PMCID: PMC2789046  PMID: 19930708
21.  Estrogen receptor beta (ERβ) subtype-specific ligands increase transcription, p44/p42 mitogen activated protein kinase (MAPK) activation and growth in human non-small cell lung cancer cells 1 
In non-small cell lung cancer (NSCLC) cells, 17β-estradiol increases transcription, activates MAPK, and stimulates proliferation. We hypothesize that estrogen receptor β (ERβ) mediates these responses because it, but not ERα, is detected in our NSCLC cell lines. To test this, we determined the effects of the ERβ-selective agonists genistein (GEN) and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) and the ERα-selective agonist 4,4’,4”-(4-propyl-[1H]-pyrazole-l,3,5-triyl)trisphenol (PPT) in 201T cells. The cells were transfected with either an ERα or an ERβ expression vector and an estrogen response element (ERE)-tk-luciferase reporter construct. PPT increased luciferase activity in cells expressing ERα but not ERβ GEN and DPN selectively increased luciferase activity in ERβ-transfected cells at concentrations ≤ 10 nM. Fulvestrant blocked the GEN and DPN-mediated increases, indicating that transcription was ER-dependent. GEN but not PPT mediated a significant 1.5-fold increase in reporter activity upon transfection with ERE-tk-luciferase alone, demonstrating that endogenous ERβ activates transcription. PPT and DPN increased MAPK phosphorylation (2.5-fold and 3.7-fold, respectively). However, only DPN stimulated 201T growth in vitro (p = 0.008) and in vivo (p = 0.05). We conclude that ERβ mediates genomic and non-genomic responses to estrogen in 201T cells and that activation of both pathways may be necessary for increased proliferation of these cells.
doi:10.1016/j.jsbmb.2009.05.004
PMCID: PMC2722836  PMID: 19460433
non-small cell lung cancer; estrogen receptor; 4,4’,4”-(4-propyl-[1H]-pyrazole-l,3,5-triyl)trisphenol; 2,3-bis(4-hydroxyphenyl)propionitrile; genistein
22.  In situ aromatase expression in primary tumor is associated with estrogen receptor expression but is not predictive of response to endocrine therapy in advanced breast cancer 
BMC Cancer  2009;9:185.
Background
New, third-generation aromatase inhibitors (AIs) have proven comparable or superior to the anti-estrogen tamoxifen for treatment of estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancer. AIs suppress total body and intratumoral estrogen levels. It is unclear whether in situ carcinoma cell aromatization is the primary source of estrogen production for tumor growth and whether the aromatase expression is predictive of response to endocrine therapy. Due to methodological difficulties in the determination of the aromatase protein, COX-2, an enzyme involved in the synthesis of aromatase, has been suggested as a surrogate marker for aromatase expression.
Methods
Primary tumor material was retrospectively collected from 88 patients who participated in a randomized clinical trial comparing the AI letrozole to the anti-estrogen tamoxifen for first-line treatment of advanced breast cancer. Semi-quantitative immunohistochemical (IHC) analysis was performed for ER, PR, COX-2 and aromatase using Tissue Microarrays (TMAs). Aromatase was also analyzed using whole sections (WS). Kappa analysis was applied to compare association of protein expression levels. Univariate Wilcoxon analysis and the Cox-analysis were performed to evaluate time to progression (TTP) in relation to marker expression.
Results
Aromatase expression was associated with ER, but not with PR or COX-2 expression in carcinoma cells. Measurements of aromatase in WS were not comparable to results from TMAs. Expression of COX-2 and aromatase did not predict response to endocrine therapy. Aromatase in combination with high PR expression may select letrozole treated patients with a longer TTP.
Conclusion
TMAs are not suitable for IHC analysis of in situ aromatase expression and we did not find COX-2 expression in carcinoma cells to be a surrogate marker for aromatase. In situ aromatase expression in tumor cells is associated with ER expression and may thus point towards good prognosis. Aromatase expression in cancer cells is not predictive of response to endocrine therapy, indicating that in situ estrogen synthesis may not be the major source of intratumoral estrogen. However, aromatase expression in combination with high PR expression may select letrozole treated patients with longer TTP.
Trial registration
Sub-study of trial P025 for advanced breast cancer.
doi:10.1186/1471-2407-9-185
PMCID: PMC2702392  PMID: 19531212
23.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987
24.  Testicular orphan nuclear receptor 4-associated protein 16 promotes non-small cell lung carcinoma by activating estrogen receptor β and blocking testicular orphan nuclear receptor 2 
Oncology Reports  2012;29(1):297-305.
The possible involvement of estrogen receptors (ERs) and testicular orphan nuclear receptors (TRs) in human non-small cell lung carcinoma (NSCLC) has been suggested, but their precise roles and their relationship remain largely unknown. This study aimed to investigate whether TR4-associated protein 16 (TRA16) regulates the ERβ and TR2 pathways and could be a potential target in NSCLC. We used tissue microarrays including NSCLC tissues (n=154) and negative controls (n=14) to examine the expression of TRA16 and ERβ, and in vitro reporter gene assays, the mammalian two-hybrid method and immunoprecipitation in Cos-1 cells to investigate the relationships among TRA16, ERβ and TR2. We found that TRA16 was highly expressed in approximately 90% of the NSCLC tissues examined. TRA16 overexpression was significantly associated with TNM stage, tumor size, lymph node metastasis, tumor thrombus in vein, tumor differentiation and prognosis of NSCLC patients, in which TRA16 was shown to be an independent prognostic factor. Introduction of TRA16 into Cos-1 cells enhanced cell proliferation. Co-expression of TRA16 and ERβ in Cos-1 cells using different reporter gene systems and mammalian two-hybrid approaches revealed that TRA16 enhanced ERβ-mediated transcriptional activity. By adopting similar approaches, and immunoprecipitation and immunocytofluorescence assays, we found that TRA16 also interacted with TR2, and blocked the TR2 inhibitory effect on ERβ. Our findings demonstrate that TRA16 could be a promising diagnostic and prognostic biomarker in NSCLC, and promotes cancer cell growth through activation of the ERβ pathway by interacting with ERβ and TR2.
doi:10.3892/or.2012.2107
PMCID: PMC3583481  PMID: 23129017
ERβ; testicular orphan nuclear receptor 2; TR4-associated protein 16; non-small cell lung cancer
25.  Prevention of tobacco carcinogen-induced lung cancer in female mice using antiestrogens 
Carcinogenesis  2012;33(11):2181-2189.
Increasing evidence shows that estrogens are involved in lung cancer proliferation and progression, and most human lung tumors express estrogen receptor β (ERβ) as well as aromatase. To determine if the aromatase inhibitor anastrozole prevents development of lung tumors induced by a tobacco carcinogen, alone or in combination with the ER antagonist fulvestrant, ovariectomized female mice received treatments with the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) along with daily supplements of androstenedione, the substrate for aromatase. Placebo, anastrozole and/or fulvestrant were administered in both an initiation and a promotion protocol of lung tumorigenesis. The combination of fulvestrant and anastrozole given during NNK exposure resulted in significantly fewer NNK-induced lung tumors (mean = 0.5) compared with placebo (mean = 4.6, P < 0.001), fulvestrant alone (mean = 3.4, P < 0.001) or anastrozole alone (mean = 2.8, P = 0.002). A significantly lower Ki67 cell proliferation index was also observed compared with single agent and control treatment groups. Beginning antiestrogen treatment after NNK exposure, when preneoplastic lesions had already formed, also yielded maximum antitumor effects with the combination. Aromatase expression was found mainly in macrophages infiltrating preneoplastic and tumorous areas of the lungs, whereas ERβ was found in both macrophages and tumor cells. Antiestrogens, especially in combination, effectively inhibited tobacco carcinogen-induced murine lung tumorigenesis and may have application for lung cancer prevention. An important source of estrogen synthesis may be inflammatory cells that infiltrate the lungs in response to carcinogens, beginning early in the carcinogenesis process. ERβ expressed by inflammatory and neoplastic epithelial cells in the lung may signal in response to local estrogen production.
doi:10.1093/carcin/bgs260
PMCID: PMC3584962  PMID: 22859269

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