Approximately 2% to 5% of endometrial cancers may be due to an inherited susceptibility. Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, an autosomal-dominant inherited cancer susceptibility syndrome caused by a germline mutation in one of the DNA mismatch repair genes, accounts for the majority of inherited cases. Lynch syndrome is associated with early onset of cancer and the development of multiple cancer types, particularly colon and endometrial cancer.
The current status of knowledge regarding Lynch syndrome-associated endometrial cancer and methods for diagnosis, screening and prevention of cancers are reviewed.
The lifetime cumulative risk of endometrial cancer for women with Lynch syndrome is 40% to 60%, which equals or exceeds their risk of colorectal cancer. No current evidence suggests either a survival advantage or disadvantage to endometrial cancer that is associated with Lynch syndrome when these cases are compared with sporadic cases. A combination of family and personal medical history and tumor testing provides an efficient combination for diagnosing Lynch syndrome in women with endometrial cancer. Current gynecologic cancer screening guidelines for women with Lynch syndrome include annual endometrial sampling and transvaginal ultrasonography beginning at age 30 to 35 years.
Diagnosing endometrial cancer patients with Lynch syndrome has important clinical implications for the individual and family members. Screening and prevention practices can decrease the likelihood of developing additional cancers.
Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription–polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1+/− = 4.91, 15-LOX-1+/+ = 3.57; WT vs 15-LOX-1+/− two-sided P = .003, WT vs 15-LOX-1+/+ two-sided P < .001; n = 10–14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.
Serous uterine cancer is not a feature of any known hereditary cancer syndrome. This study evaluated familial risk of cancers for serous uterine carcinoma patients, focusing on Lynch syndrome malignancies.
Fifty serous or mixed serous endometrial carcinoma cases were prospectively enrolled. Pedigrees were developed for 29 probands and tumors were assessed for DNA mismatch repair abnormalities. Standardized incidence ratios for cancers in relatives were estimated. A second stage analysis was undertaken using data from GOG-210. Incidence data for cancers reported in relatives of 348 serous and mixed epithelial and 624 endometrioid carcinoma patients were compared.
Nineteen of 29 (65.5%) patients in the single institution series reported a Lynch-related cancer in relatives. Endometrial and ovarian cancers were significantly over-represented and a high number of probands (6/29, 20.7%) reported pancreatic cancers. None of the probands’ tumors had DNA mismatch repair abnormalities. There was no difference in endometrial or ovarian cancer incidence in relatives of serous and endometrioid cancer probands in the case-control study. Pancreatic cancers were, however, significantly more common in relatives of serous cancer patients (OR 2.39, 95% CI 1.06–5.38).
We identified an excess of endometrial, ovarian, and pancreatic cancers in relatives of serous cancer patients in a single institution study. Follow-up studies suggest only pancreatic cancers are over-represented in relatives. DNA mismatch repair defects in familial clustering of pancreatic and other Lynch-associated malignancies are unlikely. The excess of pancreatic cancers in relatives may reflect an as yet unidentified hereditary syndrome that includes uterine serous cancers.
Endometrial cancer; Uterine serous carcinoma; Lynch syndrome; Mismatch repair; Familial clustering of cancers
HE4, also known as WFDC2, is a useful biomarker for ovarian cancer when either used alone or in combination with CA125. HE4 is also overexpressed in endometrial cancer (EC), but its function in cancer cells is not clear. In this study, we investigate the role of HE4 in EC progression. An HE4-overexpression system was established by cloning the HE4 prototypic mRNA variant (HE4-V0) into a eukaryotic expression vector. Following transfection, stable clones in two EC cell lines were selected. The effects of HE4 overexpression on cell growth and function were measured with the use of cell proliferation assay, matrigel invasion, and soft agar gel colony formation assays. HE4-induced cancer cell proliferation in vivo was examined in a mouse xenograft model. HE4 overexpression significantly enhanced EC cell proliferation, matrigel invasion, and colony formation in soft agar. Moreover, HE4 overexpression promoted tumor growth in the mouse xenograft model. HE4 overexpression enhanced several malignant phenotypes in cell culture and in a mouse model. These results are consistent with our previous observation that high levels of serum HE4 closely correlate with the stage, myometrial invasion and tumor size in patients with EC. This study provides evidence that HE4 overexpression directly impacts tumor progression in endometrial cancer.
endometrial cancer; human epididymis protein 4 (HE4); HE4 variant; proliferation; invasion; colony formation; tumorigenesis
The treatment of endometrial cancer in young women who desire future fertility poses several challenges. Oral progestin and progestin-releasing intrauterine devices (IUDs) have been shown to result in regression of endometrial hyperplasia and grade 1 endometrioid endometrial carcinoma. However, limited data are available on the use of these methods in women with grade 2 disease.
An 18 year-old nulliparous woman was diagnosed with a grade 2 endometrial adenocarcinoma. She desired future fertility and therefore underwent placement of a levonorgestrel-releasing IUD. The patient subsequently underwent endometrial sampling every 3 months, and remained disease-free 13 months after initial IUD placement.
A progestin-releasing IUD may be a valid treatment option for grade 2 endometrial cancer in young individuals who desire to retain fertility.
Tissue-based microsatellite instability analysis and immunohistochemistry for DNA mismatch repair proteins are accepted screening tools to evaluate cancer patients for Lynch Syndrome. These laboratory analyses are thus important tools in cancer prevention. Quality assurance review was performed to identify test discordances and problems. These results were then analyzed in conjunction with genetic testing outcomes. 646 consecutive tumors from 2002 to 2010 were examined. Microsatellite instability-low tumors were excluded so that 591 tumors comprised the final analyses. Discordance was defined as a discrepancy between immunohistochemistry and microsatellite instability analysis. Problem was defined as indeterminate or questionable immunohistochemistry or microsatellite instability results. All results and clinical and family histories were centrally reviewed by 2 pathologists and 1 genetics counselor. Discordances and problems were identified in 23/591 (3.9%) of the tumors. Twelve of 102 microsatellite instability-high carcinomas (11.8%) and one of 489 microsatellite stable tumors had discordant immunohistochemistry. Of these 13 tumors, 11 were from patients who had personal and/or family cancer histories concerning for a germline mismatch repair gene mutation. In addition to discordances, ten tumors with problematic immunohistochemistry profiles were identified. Accurate evaluation of microsatellite instability was possible in all tumors. In summary, concordance between immunohistochemistry and microsatellite instability was high, particularly for tumors that are microsatellite stable. Greater frequency of test discordance was identified in the tumors that were microsatellite instability-high. Thus, a major consequence of the use of immunohistochemistry by itself as a screen is the failure to identify colorectal and endometrial cancer patients who likely have Lynch Syndrome.
Lynch Syndrome; molecular testing; microsatellite instability; immunohistochemistry
Adipose tissue contains a population of tumor-tropic mesenchymal progenitors, termed adipose stromal cells (ASC), which engraft in neighboring tumors to form supportive tumor stroma. We hypothesized that intra-abdominal visceral adipose tissue may contain a uniquely tumor promoting population of ASC to account for the relationship between excess visceral adipose tissue and mortality of intra-abdominal cancers.
To investigate this, we isolated and characterized ASC from intra-abdominal omental adipose tissue (O-ASC) and characterized their effects on endometrial cancer progression as compared to subcutaneous adipose derived mesenchymal stromal cells (SC-ASC), bone marrow derived mesenchymal stromal cells (BM-MSC) and lung fibroblasts. To model chronic recruitment of ASC by tumors, cells were injected metronomically into mice bearing Hec1a xenografts.
O-ASC expressed cell surface markers characteristic of BM-MSC and differentiated into mesenchymal lineages. Co-culture with O-ASC increased endometrial cancer cell proliferation in-vitro. Tumor tropism of O-ASC and SC-ASC for human Hec1a endometrial tumor xenografts was comparable, but O-ASC more potently promoted tumor growth. Compared with tumors in SC-ASC-injected mice, tumors in O-ASC-injected mice contained higher numbers of large tortuous desmin-positive blood vessels, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. O-ASC-exhibited enhanced motility as compared to SC-ASC in response to Hec1a secreted factors.
Visceral adipose contains a population of multipotent MSC that promote endometrial tumor growth more potently than MSC from subcutaneous adipose tissue. We propose that O-ASC recruited to tumors express specific factors that enhance tumor vascularization, promoting survival and proliferation of tumor cells.
Early stage endometrial cancer is generally curable. However, progress in the treatment of advanced and recurrent endometrial cancer has been limited. This has led to a shift from the use of traditional chemotherapeutic agents and radiotherapy regimens to the promising area of targeted therapy, given the large number of druggable molecular alterations found in endometrial cancer. To maximize the effects of directed targeted therapy, careful molecular characterization of the endometrial tumor is necessary. This represents an important difference in the use of targeted therapy vs. traditional chemotherapy or radiation treatment. This review will discuss relevant pathways to target in endometrial cancer as well as the challenges that arise during development of a personalized oncology approach.
endometrial cancer; targeted therapy; personalized therapy; biomarkers; novel trial design; resistance
Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.
PTEN is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTEN sequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTEN sequencing and PTEN immunohistochemistry on each tumor. PTEN sequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, p= 0.0004). Endometrioid tumors had a significantly higher proportion of PTEN sequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTEN sequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTEN sequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (p=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTEN sequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTEN wildtype by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.
We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.
Endometrial Cancer; PTEN; PIK3CA; PIK3R1; PIK3R2
Small sample sizes used in previous studies result in a lack of overlap between the reported gene signatures for prediction of chemotherapy response. Although morphologic features, especially tumor nuclear morphology, are important for cancer grading, little research has been reported on quantitatively correlating cellular morphology with chemotherapy response, especially in a large data set. In this study, we have used a large population of patients to identify molecular and morphologic signatures associated with chemotherapy response in serous ovarian carcinoma.
A gene expression model that predicts response to chemotherapy is developed and validated using a large-scale data set consisting of 493 samples from The Cancer Genome Atlas (TCGA) and 244 samples from an Australian report. An identified 227-gene signature achieves an overall predictive accuracy of greater than 85% with a sensitivity of approximately 95% and specificity of approximately 70%. The gene signature significantly distinguishes between patients with unfavorable versus favorable prognosis, when applied to either an independent data set (P = 0.04) or an external validation set (P<0.0001). In parallel, we present the production of a tumor nuclear image profile generated from 253 sample slides by characterizing patients with nuclear features (such as size, elongation, and roundness) in incremental bins, and we identify a morphologic signature that demonstrates a strong association with chemotherapy response in serous ovarian carcinoma.
A gene signature discovered on a large data set provides robustness in accurately predicting chemotherapy response in serous ovarian carcinoma. The combination of the molecular and morphologic signatures yields a new understanding of potential mechanisms involved in drug resistance.
EphA2 overexpression predicts poor prognosis in endometrial cancer. To explore mechanisms for this association and assess its potential as therapeutic target, the relationship of EphA2 expression to markers of angiogenesis was examined using patient samples and an orthotopic mouse model of uterine cancer.
Of 85 EE C samples, EphA2 was overexpressed in 47% of tumors and was significantly associated with high VEGF expression (p = 0.001) and high MVD counts (p = 0.02). High EphA2 expression, high VEGF expression and high MVD counts were significantly associated with shorter disease-specific survival. EA5 led to decrease in EphA2 expression and phosphorylation in vitro. In the murine model, while EA5 (33–88%) and docetaxel (23–55%) individually led to tumor inhibition over controls, combination therapy had the greatest efficacy (78–92%, p < 0.001). In treated tumors, combination therapy resulted in significant reduction in MVD counts, percent proliferation and apoptosis over controls.
Expression of EphA2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was evaluated using immunohistochemistry in 85 endometrioid endometrial adenocarcinomas (EEC) by two independent investigators. Results were correlated with clinicopathological characteristics. The effect of EphA2-agonist monoclonal antibody EA5, alone or in combination with docetaxel was studied in vitro and in vivo. Samples were analyzed for markers of angiogenesis, proliferation and apoptosis.
EphA2 overexpression is associated with markers of angiogenesis and is predictive of poor clinical outcome. EphA2 targeted therapy reduces angiogenesis and tumor growth in orthotopic uterine cancer models and should be considered for future clinical trials.
endometrial cancer; EphA2; VEGF; microvessel density; angiogenesis
Assessment of estrogen receptor expression by immunohistochemistry has yielded inconsistent results as a prognostic indicator in ovarian carcinoma. In breast and endometrial carcinomas, panels of estrogen-induced genes have shown improved prognostic capability over the use of estrogen receptor immunohistochemistry alone. For both breast and endometrial cancer, over-expression of estrogen-induced genes is associated with better prognosis. We hypothesized that analysis of a panel of estrogen-induced genes can predict outcome in ovarian carcinoma and potentially differentiate between tumors of varying hormonal responsiveness. From a cohort of 219 women undergoing ovarian cancer surgery from 2004 −2007, 83 patients were selected for inclusion. All patients had advanced stage ovarian/primary peritoneal high-grade serous carcinoma and underwent primary surgical debulking followed by adjuvant treatment with platinum and taxane agents. The expression of ERα and six genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1) was measured using qRT-PCR. Unsupervised cluster analyses were used to categorize patients as high or low gene expressors. Gene expression results were then compared to those for estrogen receptor immunohistochemistry. Clusters were compared using chi-square analyses, and Cox proportional hazards were used to evaluate survival outcomes. Median follow-up time was 38.7 months (range 1–68). A cluster defined by EIG121 and ERα segregated tumors into distinct groups of high and low gene expressors. Shorter overall survival was associated with high gene expression (HR 2.84 [1.11–7.30], p=0.03), even after adjustment for other covariates. No difference in estrogen receptor immunohistochemistry expression was noted between gene clusters. In contrast to other hormonally-driven cancers, high expression of ERα and the estrogen-induced gene EIG121 predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. Such a biomarker panel may potentially be used to guide management with estrogen antagonists in this patient population.
estrogen-induced genes; ovarian high-grade serous carcinoma; hormone antagonism
The prevalence of BRCA½ mutations in germline DNA from unselected ovarian cancer patients is 11% to 15.3%. It is important to determine the frequency of somatic BRCA½ changes, given the sensitivity of BRCA-mutated cancers to poly (ADP ribose) polymerase-1 (PARP1) inhibitors and platinum analogs.
Patients and Methods
In 235 unselected ovarian cancers, BRCA½ was sequenced in 235, assessed by copy number analysis in 95, and tiling arrays in 65. 113 tumors were sequenced for TP53. BRCA½ transcript levels were assessed by quantitative polymerase chain reaction in 220. When available for tumors with BRCA½ mutations, germline DNA was sequenced.
Forty-four mutations (19%) in BRCA1 (n = 31)/BRCA2 (n = 13) were detected, including one homozygous BRCA1 intragenic deletion. BRCA½ mutations were particularly common (23%) in high-grade serous cancers. In 28 patients with available germline DNA, nine (42.9%) of 21 and two (28.6%) of seven BRCA1 and BRCA2 mutations were demonstrated to be somatic, respectively. Five mutations not previously identified in germline DNA were more commonly somatic than germline (four of 11 v one of 17; P = .062). There was a positive association between BRCA1 and TP53 mutations (P = .012). BRCA½ mutations were associated with improved progression-free survival (PFS) after platinum-based chemotherapy in univariate (P = .032; hazard ratio [HR] = 0.65; 95% CI, 0.43 to 0.98) and multivariate (P = .019) analyses. BRCA½ deficiency, defined as BRCA½ mutations or expression loss (in 24 [13.3%] BRCA½–wild-type cancers), was present in 67 ovarian cancers (30%) and was also significantly associated with PFS in univariate (P = .026; HR = 0.67; 95% CI, 0.47 to 0.96) and multivariate (P = .008) analyses.
BRCA½ somatic and germline mutations and expression loss are sufficiently common in ovarian cancer to warrant assessment for prediction of benefit in clinical trials of PARP1 inhibitors.
EphA2 is a tyrosine kinase receptor in the ephrin family that is implicated in oncogenesis and angiogenesis. Our goal was to study the role of EphA2 in endometrial cancer and its relation to steroid hormone receptor expression.
EphA2, estrogen receptor (ER), progesterone receptor (PR) and Ki-67 expression were evaluated using immunohistochemistry in 139 endometrioid endometrial carcinomas (EEC) and in 10 benign endometrial samples. Samples were scored by 2 investigators blinded to clinical outcome. Results were correlated with clinicopathologic characteristics using univariate and multivariate analysis. A p value of <0.05 was considered statistically significant.
High expression of EphA2 was detected in 48% of EEC samples vs. 10% of benign samples. EphA2 overexpression was significantly associated with high-stage (p=0.04), high-grade (p=0.003), increased depth of myometrial invasion (p=0.05), low ER (p=0.01), low PR (p=0.006) and high Ki-67 expression (p=0.04). Low ER and PR expression were both associated with high-grade, positive lymph nodes, high Ki-67 expression and high EphA2 expression. On univariate analysis of all patients, high EphA2 expression was significantly associated with shorter disease-specific survival (DSS, p<0.001). On multivariate analysis, age (p<0.001), high-stage (p=0.002) and high EphA2 expression (p=0.04) were independent predictors of poor DSS.
EphA2 overexpression is associated with aggressive phenotypic features in endometrioid endometrial carcinomas, and is inversely associated with ER and PR expression. Thus, EphA2 may be an important therapeutic target, especially in patients with hormone-receptor negative endometrial carcinoma.
Endometrial cancer; EphA2; estrogen receptor; progesterone receptor; Ki-67
To determine whether functional proteomics improves breast cancer classification and prognostication and can predict pathological complete response (pCR) in patients receiving neoadjuvant taxane and anthracycline-taxane-based systemic therapy (NST).
Reverse phase protein array (RPPA) using 146 antibodies to proteins relevant to breast cancer was applied to three independent tumor sets. Supervised clustering to identify subgroups and prognosis in surgical excision specimens from a training set (n = 712) was validated on a test set (n = 168) in two cohorts of patients with primary breast cancer. A score was constructed using ordinal logistic regression to quantify the probability of recurrence in the training set and tested in the test set. The score was then evaluated on 132 FNA biopsies of patients treated with NST to determine ability to predict pCR.
Six breast cancer subgroups were identified by a 10-protein biomarker panel in the 712 tumor training set. They were associated with different recurrence-free survival (RFS) (log-rank p = 8.8 E-10). The structure and ability of the six subgroups to predict RFS was confirmed in the test set (log-rank p = 0.0013). A prognosis score constructed using the 10 proteins in the training set was associated with RFS in both training and test sets (p = 3.2E-13, for test set). There was a significant association between the prognostic score and likelihood of pCR to NST in the FNA set (p = 0.0021).
We developed a 10-protein biomarker panel that classifies breast cancer into prognostic groups that may have potential utility in the management of patients who receive anthracycline-taxane-based NST.
Breast Cancer; Functional Proteomics; Prognosis; Prediction
Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihh acts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihh action during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgrtm2(cre)Lyd (PRcre) mouse model [Pgrtm2(cre)Lyd+Gt(ROSA)26Sortm1(Smo/EYFP)Amc+ (PRcre/+SmoM2+)]. Female PRcre/+SmoM2+ mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PRcre/+SmoM2+ uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PRcre/+SmoM2+ uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PRcre/+SmoM2+ increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.
Activation of smoothened in the mouse uterus reveals a requirement for the precise regulation of hedgehog signaling during early pregnancy and postnatal uterine development.
differentiation; implantation; mouse; smoothened; uterus
Conditional ablation of Indian hedgehog (Ihh) in the murine uterus results in mice that are sterile because of defects in embryo implantation. We performed microarray analysis on these mice at the time point at which the Ihh target genes are induced by the administration of exogenous hormone to mimic Day 3.5 of pregnancy. This analysis identified 863 genes altered by the conditional ablation of Ihh. Of these, genes that regulated the cell cycle were overrepresented. In addition, genes involved in epidermal growth factor (EGF) and estrogen (E2) signaling were found to be deregulated upon Ihh ablation. Furthermore, upon conditional ablation of Ihh, 15-mo-old mice exhibited hallmarks of estrogenized uteri, such as cystically dilated glands and hyalinized stroma. Thus, Ihh regulates embryo implantation by having an impact on the cell cycle, EGF signaling, and E2 signaling.
Gene expression profiling reveals the critical role of Indian hedgehog in cell cycle progression, EGF signaling, and the regulation of estrogen signaling in the uterus during embryo implantation.
estrogen; implantation; Indian Hedgehog; mouse; uterus
Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression, likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA = 1.78, 95% CI = 1.16–2.74, P = 0.008; and hazard ratio for TC relative to TT = 1.53, 95% CI = 1.06–2.22, P = 0.02]. Since homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome since they modify the age of colorectal cancer onset by up to 4 years.
xenobiotic metabolism; polymorphisms; Lynch syndrome; colorectal cancer; age at onset
Tuberin, the Tsc2 gene product, integrates PI3K/MAPK (mitogenic) and LKB1/AMPK (energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively-active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the NLS, and T170 was demonstrated to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress Cdk2 activity, and has been reported to mediate an anti-apoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data demonstrate that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene.
AMPK; TSC2; p27
We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese versus lean animals.
Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated pro-proliferative and anti-proliferative genes after short-term treatment with estradiol.
No significant morphological/histological difference were seen between the obese rats and the lean rats. Estrogen-induced pro-proliferative genes cyclin A and c-Myc mRNA expression were significantly higher in the endometrium of obese rats compared with that of the lean control. Expression of the anti-proliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats, however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the anti-proliferative genes RALDH2 and sFRP4 in lean rats, but had little or no effect in obese rats.
Enhancement of estrogen-induced endometrial pro-proliferative gene expression and suppression of anti-proliferative gene expression was seen in the endometrium of obese versus lean animals.
Obesity; estrogen; endometrial; proliferation
Peroxisome proliferator–activated receptor-delta (PPAR-δ) is overexpressed in human colon cancer, but its contribution to colonic tumorigenesis is controversial. We generated a mouse model in which PPAR-δ was genetically disrupted in colonic epithelial cells by targeted deletion of exon 4. Elimination of colon-specific PPAR-δ expression was confirmed by real-time reverse transcription–polymerase chain reaction (real-time RT-PCR), immunoblotting, and activity assays. Mice with and without targeted PPAR-δ genetic disruption (10–11 mice per group) were tested for incidence of azoxymethane-induced colon tumors. The effects of targeted PPAR-δ deletion on vascular endothelial growth factor expression were determined by real-time RT-PCR. Targeted PPAR-δ genetic disruption inhibited colonic carcinogenesis: Mice with PPAR-δ(−/−) colons developed 98.5% fewer tumors than wild-type mice (PPAR-δ(−/−) vs wild-type, mean = 0.1 tumors per mouse vs 6.6 tumors per mouse, difference = 6.5 tumors per mouse, 95% confidence interval = 4.9 to 8.0 tumors per mouse, P < .001, two-sided test). Increased expression of vascular endothelial growth factor in colon tumors vs normal colon was suppressed by loss of PPAR-δ expression. These findings indicate that PPAR-δ has a crucial role in promoting colonic tumorigenesis.
Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.
endometrial cancer; biomarkers; estrogen; clinical decision making; tumor recurrence