Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
The retinoblastoma gene Rb is a prototype tumor suppressor, which encodes a protein that is inactivated in a broad range of human cancers through different mechanisms. Rb functions to regulate cell proliferation, differentiation, as well as cell death. Therefore, even though Rb inactivation promotes cancer development, this may also open up certain vulnerabilities of cancers that can potentially be targeted with drug intervention. Based on the assumption that cancers that have mutation, deletion, or rearrangement in the Rb locus represent strong loss of Rb function while cancers with WT Rb on average retain some Rb function, we searched Genomics of Drug Sensitivity in Cancer database to identify cancer drugs that are particularly effective to cancers with Rb genomic alterations. Three mitotic inhibitors were identified from this analysis. We further tested the effects of two mitotic inhibitors, Taxol and STLC, on prostate and breast cancer cells. We demonstrate that the Rb status affects cancer cell sensitivity to these mitotic drugs and that the sensitizing effects of Rb are mediated in part by its regulation of the cell cycle checkpoint protein Mad2. Since the mitotic inhibitors identified in our analysis inhibit mitosis through distinct targets, it is possible that the Rb functional status may serve as a general biomarker for cancer sensitivity to mitotic inhibitors. Because the Rb pathway is inactivated in a large number of human cancers, identification of agents that are particularly effective or ineffective based on the Rb status in cancers can potentially be used generally to matching patients with appropriate treatments to achieve better therapeutic outcome.
Drug sensitivity; Rb; retinoblastoma tumor suppressor; Mad2; cell death; mitotic inhibitor; Taxol; S-Trityl-L-cysteine; STLC
Tissue hypoxia is a common pathophysiological process. Since 1990s, numerous studies have focused on investigating cellular adaptation to experimental hypoxia. A modular incubator chamber made of solid materials has frequently been used in the experiments that require hypoxic conditions. Here, we introduce a novel and inflatable chamber for hypoxia experiments. In experiments detecting hypoxia-induced accumulation of hypoxia-inducible factor 1α (HIF-1α) and hypoxia-induced expression of HIF-1-regulated genes, the new chamber yielded reproducible and comparable results as the modular incubator chamber did. The new chamber did not create inner chamber pressure during its use. Other properties of the new chamber were low-cost, easy to use, and leakage-free. Moreover, the size of the new chamber was adjustable, and the smaller one could be placed onto an inverted microscope for real-time studies. The successful examples of real-time studies included the real-time recording of GFP-HIF-1α fusion nuclear translocation and endothelial cell tubular formation.
Cell culture; hypoxia; hypoxia chamber; hypoxia-inducible factor 1
Introduction: BRCA mutations increase the risk for development of high-grade pelvic serous carcinomas. Tissue biomarkers distinguishing women at high-risk (HR) for ovarian cancer from those at low-risk (LR) may provide insights into tumor initiation pathways. Methods: A prospective study of 47 HR women (40% BRCA carriers) undergoing risk-reducing salpingo-oophorectomy and 48 LR controls undergoing salpingo-oophorectomy was performed. Ovarian/tubal tissues were harvested. Immunohistochemical analysis of candidate proteins CSF-1, CSF-1R, ErbB4 is presented, with scores separately analyzed in epithelium and stroma, in ampulla, fimbria, ovary, and ovarian endosalpingiosis (ES). Comparison was performed between HR and LR groups. Results: Elevated levels of CSF-1 (p=0.005) or ErbB4 (p=0.005) in the ovarian epithelium, or ErbB4 (p=0.005) in the ovarian stroma, were significantly associated with both the HR status and carrying a BRCA mutation, as was nuclear ErbB4 staining. Ovarian ES, an entity which likely derives from the tubal mucosal epithelium, was also associated with HR (p=0.038) and BRCA mutation status (p=0.011). Among the BRCA carriers only, markers also found association when present in the tube as well as in ovarian ES (p < 0.05). ROCs were generated including in the regression model both CSF-1 and ErbB4 expression levels. A model including CSF-1 in ovarian epithelium, ErbB4 in ovarian stroma, and younger age achieves AUC=0.87 (73% sensitivity, 93% specificity) of detection of the HR status. In BRCA carriers, CSF-1 in ovarian epithelium alone achieves AUC=0.85. Conclusions: Our data suggest that elevated levels of CSF-1/ErbB4 in the adnexae correlate with HR/BRCA carrier status. CSF-1/CSF-1R signaling is active in ovarian cancer progression; our data suggests a role in its initiation. ErbB4, in particular nuclear ErbB4, may have a role in tumor initiation as well. Ovarian ES, an entity which may represent a latent precursor to low-grade pelvic serous carcinomas, was surprisingly associated with both HR status and the BRCA carrier cohort. In line with these findings, both ErbB4 and CSF-1R expression in ovarian ES correlated with carrying a BRCA mutation. This analysis, which needs to be validated, indirectly suggests a potential link between ovarian ES and the development of pelvic serous carcinoma in women who are BRCA mutation carriers.
CSF-1; ErbB4; endosalpingiosis; high-risk
Induced pluripotent stem (iPS) cells may be a powerful tool in regenerative medicine, but their potential tumorigenicity is a significant challenge for the clinical use of iPS cells. Previously, we succeeded in converting miPS cells into cancer stem cells (CSCs) under the conditions of tumor microenvironment. Both stem cells and tumor cells are profoundly influenced by bi-directional communication with their respective microenvironment, which dictates cell fate determination and behavior. The microenvironment derived from iPS cells has not been well studied. In this paper, we have investigated the effects of secreted factors from Nanog-mouse iPS (miPS) cells on mouse Lewis lung cancer (LLC) cells that are found in the conditioned media. The results demonstrated that miPS cells secrete factors that can convert the epithelia phenotype of LLC cells to a mesenchymal phenotype, and that can promote tumorigenisity, migration and invasion. Furthermore, LLC cells that have been exposed to miPS conditioned medium became resistant to apoptosis. These various biological effects suggest that the miPS microenvironment contain factors that can promote an epithelial-mesenchymal transition (EMT) through an active Snail-MMP axis or by suppressing differentiation in LLC cells.
Mouse induced pluripotent stem cell; stem cell microenvironment; epithelial-mesenchymal transition; lung Lewis cancer cell
Raf Kinase inhibitory protein (RKIP) is a well-established metastasis suppressor that is frequently downregulated in aggressive cancers. The impact of RKIP and its phosphorylated form on disease-free survival (DFS) and other clinicopathological parameters in breast cancer is yet to be discovered. To this end, we examined RKIP expression in 3 independent breast cancer cohorts. At the Protein level, loss or reduced total RKIP expression was associated with large-sized tumors characterized by high proliferative index, high-grade and diminished estrogen (ER) and progesterone receptor expression. Loss or diminution of RKIP expression was significantly associated with shorter DFS in all cohorts. Moreover, the complete loss of p-RKIP was an independent prognostic factor using multivariate analysis in operable invasive ductal breast cancer. We show for the first time that ER, partly, drives RKIP expression through MTA3-Snail axis. Consistent with this finding, we found that, at the mRNA level, RKIP expression varied significantly across the different molecular subtypes of breast cancer with the Luminal (ER+) subtype expressing high levels of RKIP and the more aggressive Claudin-low (ER-) subtype, which depicted the highest epithelial to mesenchymal transition (EMT) registered the lowest RKIP expression levels. In conclusion, loss of expression/diminution of RKIP or its phosphorylated form is associated with poor diseases-free survival in breast cancer. Determining the expression of RKIP and p-RKIP adds significant prognostic value to the management and subtyping of this disease.
RKIP; PEBP1; ERK; estrogen receptor; aggressive cancer; breast cancer; Luminal; claudin-low; ERBB2; basal; prognosis; disease-free survival
Pancreatic cancer is the fourth leading cause of cancer related death in the US and exhibits aggressive features with short survival rate and high mortality. Therefore, it is important to understand the molecular mechanism(s) involved in the aggressive growth of pancreatic cancers, and further design novel targeted therapies for its treatment with better treatment outcome. In the present study, we found that the expression of miR-221 was significantly up-regulated in pancreatic cancer cell lines and tumor tissues compared to normal pancreatic duct epithelial cells and normal pancreas tissues. Moreover, we found that the pancreatic cancer patients with high miR-221 expression had a relatively shorter survival compared to those with lower expression, suggesting that miR-221 could be an oncogenic miRNA and a prognostic factor for poor survival of patients. Interestingly, transfection of miR-221 inhibitor suppressed the proliferative capacity of pancreatic cancer cells with concomitant up-regulation of PTEN, p27kip1, p57kip2, and PUMA, which are the tumor suppressors and the predicted targets of miR-221. Most importantly, we found that the treatment of pancreatic cancer cells with isoflavone mixture (G2535), formulated 3,3’-diindolylmethane (BR-DIM), or synthetic curcumin analogue (CDF) could down-regulate the expression of miR-221 and consequently up-regulate the expression of PTEN, p27kip1, p57kip2, and PUMA, leading to the inhibition of cell proliferation and migration of MiaPaCa-2 and Panc-1 cells. These results provide experimental evidence in support of the oncogenic role of miR-221 and also demonstrate the role of isoflavone, BR-DIM, and CDF as potential non-toxic agents that are capable of down-regulation of miR-221. Therefore, these agents combined with conventional chemotherapeutics could be useful in designing novel targeted therapeutic strategy for the treatment of pancreatic cancer for which there is no curative therapy.
miR-221; proliferation; pancreatic cancer; isoflavone; DIM; CDF
DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila.
DNA polymerase ε B subunit; Drosophila melanogaster
Notch signaling plays an essential role in development as well as cancer. We have previously shown that Notch3 is important for lung cancer growth and survival. Notch receptors are activated through the interaction with their ligands, resulting in proteolytic cleavage of the receptors. This interaction is modulated by Fringe, a family of fucose-specific β1,3 N-acetylglucosaminyltransferases that modify the extracellular subunit of Notch receptors. Studies in developmental models showed that Fringe enhances Notch’s response to Delta ligands at the expense of Jagged ligands. We observed that Manic Fringe expression is down-regulated in lung cancer. Since Jagged1, a known ligand for Notch3, is often over-expressed in lung cancer, we hypothesized that Fringe negatively regulates Notch3 activation. In this study, we show that re-expression of Manic Fringe down-regulates Notch3 target genes HES1 and HeyL and reduces tumor phenotype in vitro and in vivo. The mechanism for this phenomenon appears to be related to modulation of Notch3 protein stability. Proteasome inhibition reverses Manic Fringe-induced protein turnover. Taken together, our data provide the first evidence that Manic Fringe functions as a tumor suppressor in the lung and that the mechanism of its anti-tumor activity is mediated by inhibition of Notch3 activation.
Jagged1; manic fringe; Notch3; lung cancer
Triple-negative breast cancers (TNBCs) are heterogeneous cancers that present tumors without the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Because of the absence of these receptors, there are currently no known specific molecular targets for treatment, and although TNBC tumors are chemosensitive, prognosis is poor because this type of cancer relapses more frequently and more aggressively than hormone receptor-positive cancers. The mechanisms by which TNBCs escape control by chemotherapy are not clear, and it is crucial to identify novel molecular drivers that can be targeted in order to develop more efficient therapeutic approaches. We recently highlighted a pleiotropic role for parathyroid hormone-related protein (PTHrP) in all stages of breast cancer, and used our neutralizing anti-PTHrP monoclonal antibody (mAb M158) to efficiently inhibit progression and metastasis of human breast cancer xenografts in athymic mice. In the present study, we present evidence for a strong in vitro anti-proliferative effect of our blocking anti-PTHrP mAb M158 as a single agent on TNBC lines of various subtypes that are known to express PTHrP (MDA-MB-231, BT-549, MDA-MB-435). The same mAb is inactive in a TNBC line without detectable PTHrP expression (MDA-MB-468). In in vitro combination studies, the mAb enhances the effect of the chemotherapeutic drugs taxol and doxorubicin in PTHrP-positive TNBC cells in an additive manner. When combined with the bisphosphonate zoledronate, M158 can act in additive or antagonistic fashion in vitro depending on the cell line. Our observations identify PTHrP as a novel target against TNBC cell proliferation, and suggest that combination therapies that include an anti-PTHrP approach might increase treatment efficacy in patients with PTHrP-positive TNBC.
Breast cancer cell lines; PTHrP; TNBC; zoledronate; doxorubicin; paclitaxel; neutralizing antibody
Gene amplified in squamous cell carcinoma 1 (GASC1) is a member of Jumonji C-domain containing histone demethylases that play an essential role in affecting chromatin architecture and gene expression. The purpose of this study was to determine the expression features and the clinical significance of GASC1 in esophageal squamous cell carcinoma (ESCC). GASC1 expression was detected on tissue microarrays of ESCC samples in 185 cases using immunohistochemical staining. Strong nuclear staining for GASC1 was observed in a subset of ESCC samples. The nuclear expression of GASC1 was significantly associated with lymph node metastasis (P=0.030) and tumor-node metastasis stages (P=0.013). Kaplan-Meier survival analysis showed a tendency that high expression of GASC1 in the nucleus was associated with poor survival of ESCC patients, with a 5-year survival rate of 26.5%, as compared to 43.7% for patients with GASC1-negative/low expression. Furthermore, multivariate analysis revealed that high expression of GASC1 likely acts as a predictive factor for overall survival of ESCC patients, despite the P-value failing to reach significance (P=0.059). The findings indicate that histone demethylase GASC1 may play an important role in promoting cancer metastasis, and shed new light on the importance of targeting GASC1 to suppress metastatic disease in various tumor types, including ESCC.
Histone demethylase; GASC1; lymph node metastasis; immunohistochemistry; esophageal squamous cell carcinoma
Introduction: The ability to ascertain survival information is important for retrospective and prospective studies. Two databases that can be used are the Social Security Death Index (SSDI) and the National Death Index (NDI). Although the NDI is more complete, there are advantages to the SSDI such as ease of use and cost. The intent of this study was to determine accuracy of the SSDI. Methods: Publically available data on all known deceased individuals in the state of Ohio in 2003 were obtained from the State of Ohio Department of Health. A random sample of 63,557 of these were compared to the SSDI to identify risk factor for inclusion/exclusion. Results: Overall, 94.7% of all death records were confirmed by the SSDI. Age at death, gender, race, ethnicity, and cause of death were all found to significantly affect the likelihood of inclusion. Specifically, people aged 18-24 were included only 79.8% of the time compared to 96.2% for those over the age of 65. Also, malignancy as cause of death resulted in a 95.3% inclusion while trauma as a cause of death led to 86.5% inclusion. While Caucasians had an inclusion of 95.6%, African Americans were included only 87.8% of the time. Hispanics and women also had lower inclusion rates. Discussion: The SSDI is a strong tool for following up on participants lost to follow up in certain populations but is weaker in others. The SSDI would be particularly useful in a population that is largely older, Caucasian, or has malignant disease.
Social; security; death; index; SSDI; NDI; survival
Background: Tumor-associated macrophages (TAMs) are a key component of the inflammatory microenvironment. Their role in prostate cancer development and progression remains unclear. We examined whether the amount of TAMs in prostate cancer is: 1) higher than prostatic intraepithelial neoplasia (PIN) and benign tissue 2) associated with poorly differentiated disease, and 3) predictive of biochemical recurrence among surgically treated men. Methods: A tissue microarray (TMA) of prostatectomy specimens from 332 patients was stained for CD68, a TAM marker. A separate TMA was used for validation. Associations between mean TAMs in cancer cores and PSA recurrence were determined by Cox proportional hazards models after adjusting for age, preoperative PSA, race, body mass index, pathologic Gleason sum, seminal vesicle invasion, extracapsular extension, and margin status. Results: Mean TAM number was higher in cancer versus PIN and benign tissue (p<0.0001). Mean TAM number was higher in Gleason grade 4 cores vs. Gleason grade 3 cores (p=0.003). On multivariable analysis, no association was observed between mean TAM number per cancer core and biochemical recurrence in either cohort. Conclusion: Mean TAM number was higher in cancer cores vs. PIN and benign tissue, and higher in high grade prostate cancer supporting the potential role of TAMs in prostate cancer development. However, TAMs were not associated with biochemical recurrence after radical prostatectomy suggesting TAM counts do not provide independent prognostic value among surgically treated men. Further studies are required to elucidate the functional significance of TAMs in the prostate cancer microenvironment.
Biochemical recurrence; cancer development; prostate; tumor associated macrophages; tissue microarray
Our group recently demonstrated in a rat model that pretreatment with morphine facilitates doxorubicin delivery to the brain in the absence of signs of increased acute systemic toxicity. Morphine and other drugs such as dexamethasone or ondansetron seem to inhibit MDR proteins localized on blood-brain barrier, neurons and glial cells increasing the access of doxorubicin to the brain by efflux transporters competition. We explored the feasibility of active modification of the blood-brain barrier protection, by using morphine dexamethasone or ondansetron pretreatment, to allow doxorubicin accumulation into the brain in a rodent model. Rats were pretreated with morphine (10 mg/kg, i.p.), dexamethasone (2 mg/kg, i.p.) or ondansetron (2 mg/kg, i.p.) before injection of doxorubicin (12 mg/kg, i.p.). Quantitative analysis of doxorubicin was performed by mass spectrometry. Acute hearth and kidney damage was analyzed by measuring doxorubicin accumulation, LDH activity and malondialdehyde plasma levels. The concentration of doxorubicin was significantly higher in all brain areas of rats pretreated with morphine (P < 0.001) or ondansetron (P < 0.05) than in control tissues. The concentration of doxorubicin was significantly higher in cerebral hemispheres and brainstem (P < 0.05) but not in cerebellum of rats pretreated with dexamethasone than in control tissues. Pretreatment with any of these drugs did not increase LDH activity or lipid peroxidation compared to controls. Our data suggest that morphine, dexamethasone or ondansetron pretreatment is able to allow doxorubicin penetration inside the brain by modulating the BBB. This effect is not associated with acute cardiac or renal toxicity. This finding might provide the rationale for clinical applications in the treatment of refractory brain tumors and pave the way to novel applications of active but currently inapplicable chemotherapeutic drugs.
Doxorubicin; morphine; dexamethasone; ondansetron; blood-brain barrier; rodent model; MDR transporters; mass spectrometry
The properties of stem cells can be induced during the epithelial to mesenchymal transition (EMT). The responsible molecular mechanisms, however, remain largely undefined. Here we report the identification of the microRNA-146a (miR-146a) as a common target of Krüppel-like factor 8 (KLF8) and TGF-β, both of which are known EMT-inducers. Upon KLF8 overexpression or TGF-β treatment, a significant portion of the MCF-10A cells gained stem cell traits as demonstrated by an increased expression of CD44high/CD24low, activity of aldehyde dehydrogenase (ALDH), mammosphere formation and chemoresistance. Along with this change, the expression of miR-146a was highly upregulated in the cells. Importantly, we found that miR-146a was aberrantly co-overexpressed with KLF8 in a panel of invasive human breast cancer cell lines. Ectopic expression of KLF8 failed to induce the stem cell traits in the MCF-10A cells if the cells were pre-treated with miR-146a inhibitor, whereas overexpression of miR-146a in the MCF-10A cells alone was sufficient to induce the stem cell traits. Co-staining and luciferase reporter analyses indicated that miR-146a targets the 3’-UTR of the Notch signaling inhibitor NUMB for translational inhibition. Overexpression of KLF8 dramatically potentiated the tumorigenecity of MCF-10A cells expressing the H-Ras oncogene, which was accompanied by a loss of NUMB expression in the tumors. Taken together, this study identifies a novel role and mechanism for KLF8 in inducing pro-tumorigenic mammary stem cells via miR-146a potentially by activating Notch signaling. This mechanism could be exploited as a therapeutic target against drug resistance of breast cancer.
KLF8; miR-146a; EMT; mammary stem cells; tumorigenesis
We have identified an alternatively spliced, non-functional aberrant E-cadherin transcript that lacks exon 11 and is over expressed in malignant cells as compared to the normal non-malignant cells. This increase in the aberrant transcript is a mechanism of loss of E-cadherin gene expression as it is rapidly degraded by the nonsense mediated decay pathway. To study the mechanism of this gene missplicing we analyzed the role of histone epigenetic modifications in lung cancer cell lines. The treatment of low E-cadherin lung cancer cell lines with histone deacetylase inhibitor (HDACi, MS-275) resulted in the preferential expression of the correctly spliced transcripts in the low E-cadherin expressing cell lines only. Chromatin immunoprecipitation (ChIP) assays revealed that the histone hypoacetylation levels correlate with aberrant exon 11 splicing as there is more aberrant splicing in cell lines with E-cadherin promoter hypoacetylation. Inactivation of histone deacetylases (HDAC) 1, 2 and 3 resulted in an increase in E-cadherin expression and an increase in the ratio of the correctly spliced E-cadherin transcript. As transcription of the gene is closely linked to splicing, we considered the possibility that change in E-cadherin transcription correlates with splicing. The Zeb1 epithelial-mesenchymal transformation (EMT) inducer silences E-cadherin expression and could also alter the splicing of this exon. Inhibition of the E-cadherin promoter transcription with Zeb1 expression increases aberrant splicing and the reverse is observed when Zeb1 is knocked down. The role of HDAC inhibitors was also studied in vivo in a immunodeficient mouse xenograft model. Exposure of mice to HDACi resulted in growth inhibition, increase in E-cadherin expression, alteration of aberrant splicing and the reversal of EMT in mouse tumors. The findings support the modulation of E-cadherin exon 11 inclusion or exclusion by histone epigenetic modifications as they change the overall chromatin structure. The results provide an interesting link between epigenetic alterations in cancer cells and gene splicing in addition to their effect on gene silencing.
E-cadherin; splicing; histone modifications; HDAC; HDAC inhibitor; Zeb1; EMT
Historically, metastatic renal cell carcinoma (mRCC) is more resistant to conventional cytotoxic chemotherapeutic agents than other solid tumors. Although significant progress has been made over the last decade with several novel therapeutics, these agents invariably go on to fail, largely due to either intrinsic or acquired resistance. To help overcome, or at least delay resistance, combinatorial therapies utilizing agents with disparate, and ideally complementary, mechanisms of actions are needed. In this report, we assess the novel combination of the mTOR inhibitor, temsirolimus, with the microtubule stabilizing drug ixabepilone in RCC. Our results demonstrate synergy in multiple cell lines of RCC and further evaluation of this combination is warranted in the clinical setting. Activation of the endoplasmic reticulum (ER) stress response pathway may in part explain the combinatorial synergy. We further propose that ER stress induced proteins may serve as early response biomarkers to combinatorial therapy in a clinical trial.
Renal cell carcinoma; ixabepilone; microtubule stabilizer; mTOR inhibitor; temsirolimus; combination therapy
In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.
Expression microdissection; esophageal squamous cell carcinoma; RNAscope; immunoblot
Background: malignant peritoneal mesothelioma (MPM) is a rare peritoneal mesothelial neoplasm. Ki67 and BCL2 are established prognostic markers in several cancers. High Ki67 expression indicates tumour progression, whilst similar expression of BCL2 retards tumour replication. Traditionally, prognosis in MPM is gauged with a single biomarker assessed separately in a dichotomous manner. Here, we examine prognosis with dual biomarkers incorporated in a model to predict survival. Materials and methods: Forty two MPM archival patient tumours were screened for Ki67 and BCL2 by immunohistochemistry and evaluated using standard methods. Ki67 and BCL2 expression was incorporated into a prognostic model to develop Ki67-BCL2 index. Using this index, three hazard groups were identified (high, medium and low risk). Kaplan-Meier survival analysis was performed to assess the significance of these hazard groups in the various clinicopathological categories. Results: In all clinicopathological categories, high risk group showed poor prognosis compared to low risk group (p = < 0.001). Compared to medium risk, high risk group carried poor prognosis in all tumours, females, epitheloid tumours, peritoneal cancer index (PCI) < 20, ≥ 20, age at diagnosis (AAD) < 60, and ≥ 60 years. Independent of the Ki67-BCL2 index, male, sarcomatoid, PCI ≥ 20 and AAD ≥ 60 were poor prognostic factors. High risk group was an independent poor prognostic factor in all tumours, males, females and age < 60 years. The distribution of high risk: low risk group in male and female was 3: 2 and 2: 3, respectively, indicating a gender difference. Comparing hazard ratios generated by Ki67-BCL2 index to that of either Ki67 or BCL2, as a single prognostic biomarker, there was a reduction of HR values. Conclusion: Ki67-BCL2 index seems to suggest a more sensitive method of predicting prognosis. However, the current model needs further evaluation in an independent large cohort sample.
Ki67; BCl2; prognosis; malignant peritoneal mesothelima
Arginine is one of the essential amino acid involved in numerous biosynthetic pathways that significantly influence tumor growth. It has been demonstrated that arginine is effective to inhibit proliferation of cancer cells when an appropriate dose is applied. Generally, induction of cell death requires high concentration of arginine while low concentration of arginine facilitates cell proliferation. In addition to the apoptosis induced by metabolism of arginine, it has also been reported that in an ideal solution environment, arginine may assemble into arginine clusters to kill cancer cells. Therefore, to make the arginine an effective anticancer agent, arginine/albumin microspheres were designed and synthesized to provide a localized high concentration of arginine on tumor sites. In addition, the arginine/albumin mesospheres (AAMS) are also expected to provide an arginine-rich surface on microspheres, which is similar to the arginine cluster, to effectively inhibit tumor growth. In this study, the AAMS were synthesized through a water/organic solvent emulsion system and the surface properties were characterized. The in vitro effects of AAMS on A549, CRL-2081, MAK9 lung cancer cells (LCC) proliferation, migration, and tumor growth were determined. The expression of oncogenic protein EphA2 and transcription factor slug was also determined. AAMS significantly inhibited the cell proliferation, cell migration and tumor growth in all the three LCC, while same concentration of free arginine promoted the LCC tumor growth and migration. Our studies indicate that the synthesized AAMS has a more effective inhibiting effect on proliferation, migration and tumor growth of LCC than freely released arginine. The expression of EphA2 receptor mRNA was significantly decreased when compared to control cells. In addition the mRNA expression of transcription factor slug was also inhibited by AAMS suggesting that AAMS affects the expression of EphA2 and slug and may regulate LCC proliferation and migration. These data suggests that the AAMS can be an ideal delivery vehicle for therapeutic interventions against LCCs.
Arginine-conjugated albumin; microspheres; inhibition; proliferation; migration; lung cancer
A gene family expressed in prostate, ovary, testis and placenta (POTEs) is newly defined and primate-specific. POTE genes have 13 paralogs, which are dispersed in 8 chromosomes and divided into three groups. The proteins encoded by these genes contain three domains: An N-terminal, ankyrin repeats and a C-terminus. Previous studies suggest that POTE proteins are localized in the inner aspect of cellular membrane and are considered as cancer-testis antigens, because they expressed widely in cancers, but in limited benign tissues. In this study, we will study the subcellular distribution of all POTE proteins and their associations with the progress and metastasis of malignancies. By performing Immunohistochemistry, Immunocytochemistry and immunofluorescence assay on tissue microarray slides containing tissues with different pathology and origins or on cell lines, we found that the epitopes of N- and C-terminals of all detected POTEs were widely expressed in benign and malignant tissues. Among these epitopes, C-terminal common to group 3 POTEs (CtG3P) was the only portion localized in nucleoli. The nucleolar IHC scores for CtG3P was lowest in benign tissues (4.47 ± 3.43), significantly higher in localized malignancies (5.32 ± 3.36, p = 3.63E-02), and highest in metastatic malignancies (7.90 ± 2.29, p = 8.13E-12). The CtG3P was better in differentiation of benign from malignant changes, and/or in differentiation of localized from metastatic cancers as compared with Ki-67 and AgNORs. In addition, transient transfection of siRNA against mRNA of group 3 POTEs influences the growth and survival of MCF-7 cells in vitro in a dose dependent manner.
POTEs gene family; prostate; ovary; testis and placenta; cancer; ankyrin repeats; cancer-testis antigens; nucleolar marker; malignant progression; metastasis
p140Cap is an adaptor protein that negatively controls tumor cell properties, by inhibiting in vivo tumor growth and metastasis formation. Our previous data demonstrated that p140Cap interferes with tumor growth and impairs invasive properties of cancer cells inactivating signaling pathways, such as the tyrosine kinase Src or E-cadherin/EGFR cross-talk. In breast cancer p140Cap expression inversely correlates with tumor malignancy. p140Cap is composed of several conserved domains that mediate association with specific partners. Here we focus our attention on two domains of p140Cap, the TER (Tyrosine Enriched Region) which includes several tyrosine residues, and the CT (Carboxy Terminal) which contains a proline rich sequence, involved in binding to SH2 and SH3 domains, respectively. By generating stable cell lines expressing these two proteins, we demonstrate that both TER and CT domains maintain the ability to associate the C-terminal Src kinase (Csk) and Src, to inhibit Src activation and Focal adhesion kinase (Fak) phosphorylation, and to impair in vitro and in vivo tumor cell features. In particular expression of TER and CT proteins in cancer cells inhibits in vitro and in vivo growth and directional migration at a similar extent of the full length p140Cap protein. Moreover, by selective point mutations and deletion we show that the ability of the modules to act as negative regulators of cell migration and proliferation mainly resides on the two tyrosines (Y) inserted in the EPLYA and EGLYA sequences in the TER module and in the second proline-rich stretch contained in the CT protein. Gene signature of cells expressing p140Cap, TER or CT lead to the identification of a common pattern of 105 down-regulated and 128 up-regulated genes, suggesting that the three proteins can act through shared pathways. Overall, this work highlights that the TER and CT regions of p140Cap can efficiently suppress tumor cell properties, opening the perspective that short, defined p140Cap regions can have therapeutic effects.
p140Cap; breast cancer; lung cancer; colon cancer; cell signaling; Csk; Src
Association studies suggest that thyroid hormone receptor β (TRβ) could function as a tumor suppressor in breast cancer development, but unequivocal evidence is still lacking. To understand the role of TRβ in breast tumor development, we adopted the gain-of-function approach by stably expressing the THRB gene in a human breast cancer cell line, MCF-7 (MCF-7-TRβ). Parental MCF-7 cells express the estrogen receptor, but not TRs. MCF-7 cells, stably expressing only the selectable marker, the Neo gene, were also generated as control for comparison (MCF-7-Neo cells). Cell-based studies indicate that the estrogen (E2)-dependent growth of MCF-7 cells was inhibited by the expression of TRβ in the presence of the thyroid hormone (T3). In a xenograft mouse model, large tumors rapidly developed after inoculation of MCF-7-Neo cells in athymic mice. In contrast, markedly smaller tumors (98% smaller) were found when MCF-7-TRβ cells were inoculated in athymic mice, indicating that TRβ inhibited the E2-dependent tumor growth of MCF-7 cells. Further detailed molecular analysis showed that TRβ acted to activate apoptosis and decrease proliferation of tumor cells, resulting in inhibition of tumor growth. The TRβ-mediated inhibition of tumor growth was elucidated via down-regulation of the JAK-STAT-cyclin D pathways. This in vivo evidence shows that TRβ could act as a tumor suppressor in breast tumorigenesis. The present study provides new insights into the role of TR in breast cancer.
Thyroid hormone receptor beta; tumor suppressor; tumorigenesis; STAT signaling; MCF-7 cells
Background Malignant peritoneal mesothelioma (MPM) is a rare neoplasm of the peritoneal membrane that is causally related to asbestos exposure. Survival after treatment is poor. Current therapy involving hyperthermic intraperitoneal chemotherapy has improved survival in selective patients. In the past, several prognostic factors have been identified in MPM patients and this has prompted the development of new therapies and patient management. Since BCL2, an antiapoptotic oncoprotein, is a favourable prognostic factor in breast cancer, we investigated to determine the significance of BCL2 in MPM. Materials and Methods Forty two archival patient tumour sections embedded in paraffin blocks were sectioned and subjected to immunohistochemistry to detect BCL2. The staining intensity and abundance was classified using standard procedures and classified into two groups (0-4 = low & 5-8 = high expression). The distribution of BCL2 groups was examined in the different clinicopathological categories to determine prognosis using Kaplan–Meier survival analysis. Results: Univariate analysis revealed that in almost all clinicopathological categories, high BCL2 expression predisposed patients to a favourable prognosis. Independent of BCL2 expression, univariate analysis also showed that male gender, sarcomatoid histology, high PCI and age at diagnosis ≥ 60 years were associated poor prognosis. Multivariate analysis indicated that for all tumours, males and females, high BCL2 expression was associated with good prognosis. Further, independent of BCL2, age ≥ 60 years is an unfavourable prognostic factor. Conclusion: Expression of BCL2 may serve to distinguish prognosis within the individual clinicopathological categories. BCL2 is also an independent variable in all tumours, males and females, with high expression being associated with good prognosis.
BCL2; prognosis; peritoneal mesothelioma; survival
Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m2/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include pre-existing neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m2/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).
Rigosertib; ON01910.Na; phase 1 study; polo-like kinase; phosphatidylinositol-3-kinase