George Larry Maxwell

Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F

Class 3 semaphorins (SEMA), SEMA3B and SEMA3F, are secreted proteins that regulate angiogenesis, tumor growth, and metastasis by binding to their transmembrane receptor complex consisting of plexins and neuropilins (NP). Expression of SEMAs and their receptors was assessed in tissue microarrays by immunohistochemistry. SEMA3B, SEMA3F, and plexin A3 were expressed strongly in normal endometrial tissues, whereas grade-dependent decreases were found in endometrial carcinomas. No change was observed in the expression of plexin A1, NP1, and NP2 in normal versus endometrial cancer tissues. Endometrial cancer cells showed decreased expression of SEMA3B, SEMA3F, and plexin A3 compared with their normal counterparts. Treatment of cancer cells with progesterone (P4) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for a period of 72 hours induced a significant upregulation of SEMA3B and SEMA3F as well as inhibited growth of cancer cells by increasing caspase-3 activity. Cotreatment of cell lines with P4 or 1,25(OH)2D3 and their respective antagonists confirmed the specificity of their actions. Transfection of siRNA-targeting SEMA3B and SEMA3F in endometrial cancer cells attenuated P4 or 1,25(OH)2D3-induced growth inhibition. Restoration of SEMA3B or SEMA3F expression in cancer cells caused growth inhibition, reduced soft agar colony formation, and cell invasiveness by inhibiting expression of matrix metalloproteinase-2 (MMP-2), MMP-9, integrin αvβ3, and proangiogenic genes and by upregulating antiangiogenic genes. Thus, we have identified two new P4 and 1,25(OH)2D3-regulated antitumor genes for endometrial cancer. These results suggest that the loss of SEMAs contribute to the malignant phenotype of endometrial cancer cells and that reexpression of SEMAs by ectopic expression or with anticancer agents P4 or 1,25(OH)2D3 can be a promising therapeutic treatment against endometrial cancer. Mol Cancer Res; 9(11); 1479–92. ©2011 AACR.

Progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells.

Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0–G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry–based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2–associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell-cycle arrest. Cancer Prev Res; 6(7); 731–43. ©2013 AACR.

Progestin Treatment Induces Apoptosis and Modulates Transforming Growth Factor-β in the Uterine Endometrium

Background: Epidemiologic, animal, and human data suggest that progestins are potent endometrial cancer preventive agents. In the ovarian surface epithelium, progestins have been hypothesized to confer a cancer preventive effect via apoptosis and modulation of transforming growth factor-β (TGF-β). Given that the ovarian epithelium and endometrium share a common embryologic origin and similar reproductive and hormonal risk factors for malignancy, we tested the hypothesis that progestins confer biological effects in the endometrium similar to those in the ovary. Methods: Postmenopausal female macaques (n = 78) were randomized into four groups to receive a diet for 36 months containing no hormone versus conjugated equine estrogen (CEE), medroxyprogesterone acetate (MPA), or CEE + MPA. The endometrium was then examined immunohistochemically for treatment-specific changes using antibodies to activated caspase-3 (for apoptosis), Ki-67 (proliferation), and the TGF-β1, TGF-β2, and TGF-β3 isoforms. Results: Percentages of caspase-positive endometrial glandular cells were 3- to 5-fold higher in CEE + MPA–treated animals compared with all others (P < 0.05). Caspase-expressing cells were six times more numerous in the endometrial stroma of animals treated with MPA alone relative to other groups (P < 0.0001). Induction of endometrial glandular cell apoptosis in the CEE + MPA–treated group was associated with a dramatic increase in expression of TGF-β2 and TGF-β3 in the stromal compartment of the endometrium (P < 0.0001). Conclusion: Progestin treatment activates chemopreventive biological effects in the endometrium that are similar to those in the ovarian surface epithelium. These data may facilitate identification of a chemopreventive approach that dramatically lessens the risk of both uterine and ovarian cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(3):578–84)

Identifier mapping performance for integrating transcriptomics and proteomics experimental results

BackgroundStudies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit.ResultsWe compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed.ConclusionsThe methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for omic data merging.

Assessment of the Effects of Severe Obesity and Lifestyle Risk Factors On Stage of Endometrial Cancer

Background: Lifestyle risk factors, including obesity, have been associated with increased risk of endometrial cancer (EC). Women with higher obesity levels tend to have less aggressive EC disease stage and histology. This study further investigated associations between nonmodifiable risk factors, such as age, race, and grade, and modifiable lifestyle factors, such as diet and physical activity expenditure, in relation to severe obesity and late versus early EC stage at diagnosis. Methods: Demographic, anthropometric, and lifestyle surveys were administered to 177 women with histologically confirmed EC. Logistic regression analyses assessed the relationship between obesity and other risk factors on EC stage at diagnosis. Results: In multivariate models, body mass index (BMI) < 35 was not significantly associated with late EC stage at diagnosis (OR = 1.67, P = 0.219) when adjusting for grade and age. Grade was significantly associated with EC stage when controlling for BMI and age (OR = 8.48, P = .000). Women more than the age of 60 had a fourfold increased risk of diagnosis at late versus early EC stage when adjusting for other risk factors. Age had a confounding effect on the obesity-EC stage association. Conclusions: Our results corroborate those of past studies showing that BMI is not an independent risk factor for EC stage and that age may have confounded the obesity-EC stage association. Because of mixed results and implications for treatment outcomes, however, further research examining these variables is warranted. Impact: Our results provide further insight into the obesity EC-stage association, especially the confounding effect of age. Future studies should examine modifiable lifestyle factors in larger and more diverse populations. Cancer Epidemiol Biomarkers Prev; 22(1); 76–81. ©2012 AACR.

Evidence of a Chemopreventive Effect of Progestin Unrelated to Ovulation on Reproductive Tract Cancers in the Egg-laying Hen

Epidemiologic, laboratory, and animal evidence suggests that progestins and vitamin D may be potent ovarian cancer preventives. Our objectives were to evaluate progestins as reproductive tract cancer chemopreventives in the chicken, determine whether restricted ovulation affected the incidence of reproductive tract tumors, and assess whether vitamin D would confer cancer protection either alone or in addition to progestin. A total of 2,400 two-year-old Single Comb White Leghorns were randomized into six groups (400 each) with hormonal and dietary manipulation for 2 years as follows: (i) no intervention, regular feed/caloric intake, (ii) control, (iii) vitamin D, (iv) the progestin levonorgestrel, (v) vitamin D plus levonorgestrel, and (vi) the progestin Provera (medroxyprogesterone acetate). Groups 2 to 6 were caloric restricted to inhibit ovulation. Our results indicated that caloric restriction decreased egg production by more than 60%, and was associated with a greater than 70% decrease in reproductive tract cancers. Ovulatory events did not differ among the caloric-restricted groups (groups 2–6), except for the group receiving levonorgestrel, which had fewer ovulatory events than controls (P = 0.046). After correcting for egg production, birds receiving progestins had significantly fewer reproductive tract cancers [OR, 0.61; confidence interval (CI), 0.39–0.95; P = 0.03], with similar proportionate reductions in tumors arising in either the ovary or oviduct. Vitamin D did not significantly affect cancer incidence overall, or add to the cancer preventive effect of progestins. This study suggests a protective effect of progestins against ovarian and oviductal cancers. These data support the concept that progestins provide a chemopreventive effect unrelated to ovulation. Cancer Prev Res; 6(12); 1283–92. ©2013 AACR.

Racial disparities in risk of second primary cancers in endometrial cancer patients: Analysis of SEER Data.

Introduction: Endometrial cancer (EC) is the most common gynecologic cancer in the United States. Racial disparities in the incidence and mortality of this cancer are apparent; black women are less likely to develop this malignancy and yet are more likely to die when diagnosed. Racial differences of second primary cancer (SPC) have not been examined, and the goal of this study was to examine these differences. Methods: With the use of the National Cancer Institutes Surveillance, Epidemiology, and End Results database, SPC risk in white patients and black patients with EC was compared to the general population and to women with other primary cancers. Standardized incidence ratios (SIRs) of SPC (overall and by tumor site) with 95% confidence intervals were calculated. Poisson regression was used to estimate the race-specific risk of SPC in EC cases treated with radiotherapy versus nonirradiated cases. Results: The analysis included 11,047 patients with EC diagnosed between 1973 and 2007 that developed an SPC. Overall risk of SPC in white women with EC was significantly lower than that in the general population (SIR = 0.85; 95% CI, 0.84-0.87) but significantly higher in black women with EC (SIR = 1.19; 95% CI, 1.08-1.31). White women with EC treated with radiotherapy were more likely to develop SPC compared with nonirradiated cases (incidence rate ratio [IRR], 1.18; 95% CI, 1.14-1.23). Conclusions: This is the first analysis of race-specific SPC risk in EC cases, and it suggests differences between white women and black women with EC. Although exploratory, these data provide important clues about the etiology of SPC in patients with EC. This analysis also highlights the need for careful monitoring after diagnosis and treatment of EC.

Interim analysis of a phase I/IIa trial assessing E39+GM-CSF, a folate binding protein vaccine, to prevent recurrence in ovarian and endometrial cancer patients

BACKGROUND Folate binding protein(FBP) is an immunogenic protein over-expressed in endometrial(EC) and ovarian cancer(OC). We are conducting a phase I/IIa trial of E39 (GALE 301)+GM-CSF, an HLA-A2-restricted, FBP-derived peptide vaccine to prevent recurrences in disease-free EC and OC patients. This interim analysis summarizes toxicity, immunologic responses, and clinical outcomes to date. METHODS HLA-A2+ patients were vaccinated(VG), and HLA-A2- or -A2+ patients were followed as controls(CG). Six monthly intradermal inoculations of E39+250mcg GM-CSF were administered to VG. Demographic, safety, immunologic, and recurrence rate(RR) data were collected and evaluated. RESULTS This trial enrolled 51 patients; 29 in the VG and 22 in the CG. Fifteen patients received 1000mcg E39, and 14 received <1000mcg. There were no clinicopathologic differences between groups(all p = 0.1). E39 was well-tolerated regardless of dose. DTH increased pre- to post-vaccination (5.71.5 mm vs 10.33.0 mm, p = 0.06) in the VG, and increased more in the 1000mcg group (3.82.0 mm vs 9.53.5 mm, p = 0.03). With 12 months median follow-up, the RR was 41% (VG) vs 55% (CG), p = 0.41. Among the 1000mcg patients, the RR was 13.3% vs 55% CG, p = 0.01. Estimated 2-year DFS was 85.7% in the 1000mcg group vs 33.6% in the CG (p = 0.021). CONCLUSIONS This phase I/IIa trial reveals that E39+GM-CSF is well-tolerated and elicits a strong, dose-dependent in vivo immune response. Early efficacy results are promising in the 1000 mcg dose cohort. This study proves the safety and establishes the dose of E39 for a larger prospective, randomized, controlled trial in HLA-A2+ EC and OC patients to prevent recurrence.

Progesterone potentiates the growth inhibitory effects of calcitriol in endometrial cancer via suppression of CYP24A1

Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination. Progestins inhibited calcitriol-induced expression of CYP24A1 and splice variant CYP24SV mRNA and protein in cancer cells. Furthermore, actinomycin D, but not cycloheximide, blocked calcitriol-induced CYP24A1 splicing. siRNA-induced knockdown of CYP24A1 expression sensitized endometrial cancer cells to calcitriol-induced growth inhibition. These data suggest that CYP24A1 overexpression reduces the antitumor effects of calcitriol in cancer cells and that progestins may be beneficial for maintaining calcitriols anti-endometrial cancer activity.

Racial disparities in molecular subtypes of endometrial cancer

OBJECTIVESnRacial differences in the molecular subtypes of endometrial cancer and associations with progression-free survival (PFS) were evaluated.nnnMETHODSnMolecular, clinical and PFS data were acquired from the Cancer Genome Atlas (TCGA) including classification into the integrative, somatic copy number alteration and transcript-based subtypes. The prevalence and prognostic value of the aggressive molecular subtypes (copy number variant [CNV]-high, cluster 4 or mitotic) were evaluated in Black and White patients.nnnRESULTSnThere were 337 patients including 14% self-designated as Black, 27% with advanced stage, and 82% with endometrioid histology. The CNV-high subtype was more common in Black than White patients (61.9% vs. 23.5%, P=0.0005) and suggested worse PFS in Black patients (hazard ratio [HR]=3.4, P=0.189). The cluster 4 subtype was more prevalent in Black patients (56.8% vs. 20.9%, P<0.0001) and associated with worse PFS in Black patients (HR=3.4, P=0.049). The mitotic subtype was more abundant in Black patients (64.1% vs. 33.7%, P=0.002), indicated worse PFS in Black patients (HR=4.1, P=0.044) including the endometrioid histology (HR=6.1, P=0.024) and exhibited race-associated enrichment in cell cycle signaling and pathways in cancer including PLK1 and BIRC7. All of these aggressive molecular subtypes also indicated worse PFS in White patients, with unique enrichments in mitotic signaling different from Black patients.nnnCONCLUSIONSnThe aggressive molecular subtypes from TCGA were more common in Black endometrial cancer patients and indicated worse PFS in both Black and White patients. The mitotic subtypes also indicated worse PFS in Black patients with endometrioid histology. Enrichment patterns in mitotic signaling may represent therapeutic opportunities.