Cristina Mirantes

Epithelial-to-mesenchymal transition and stem cells in endometrial cancer

This review article describes the main features of epithelial-to-mesenchymal transition (EMT) and its possible role in understanding myometrial invasion in endometrial carcinoma (EC), as well as the development of malignant mixed Müllerian tumor (MMMT). Moreover, the article discusses the possible role of somatic (SSC) and cancer stem cells (CSC) in EC. Different transcriptional repressors of E-cadherin have been identified in EMT, including Snail and Slug, ZEB1 and ZEB2, and E47 and Twist. The expression of some of these genes is increased at the myoinvasive front and correlates inversely with E-cadherin inmunoreactivity. Whereas the transient occurrence of the EMT phenomenon is important for myometrial invasion in conventional EC, MMMT shows permanent expression of EMT leading to repression of E-cadherin and increased expression of mesenchymal markers including proteins involved in skeletal muscle development. An SSC population, identified as a side population, assessed by the Hoechst dye exclusion test has been identified in human endometrium. CSCs have been defined in analogy to SSC as cancer cells that have the capacity to self-renew, which means undergoing divisions that allow the generation of more identical CSCs and give rise to the variety of more differentiated cells found in the malignancy. Although published data show that CD133(+) cells retain the characteristics of CSC, there is no conclusive evidence showing that CD133 is the universal marker for EC stem cells. Finally, a possible role for endometrial stem cells in the development of ovarian endometriosis and ovarian endometrioid carcinoma is commented.

KSR1 Is Overexpressed in Endometrial Carcinoma and Regulates Proliferation and TRAIL-Induced Apoptosis by Modulating FLIP Levels

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues

In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P < .05) and included a high target retrieval solution, 1:100 primary antibody dilution (2.925 mg/L), FLEX diluent, and EnVisionFLEX+ detection method, with a sensitivity and specificity of 72.33% and 78.57%, respectively. Staining specificity was confirmed in cell lines and animal models. Endometrial carcinomas with PTEN genetic abnormalities showed statistically lower staining than tumors without alterations (mean histoscores, 34.66 and 119.28, respectively; P = .01). Controlled preanalytical factors (delayed fixation and overfixation) did not show any statistically significant effect on staining with optimal protocol (P > .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01).

Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin.

Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis.

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

SUMMARY PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

Stem cells in human endometrium and endometrial carcinoma.

This study reviews the current knowledge about endometrial somatic stem cells and endometrial cancer stem cells. It describes the main features of somatic stem cells, such as high proliferative potential, self renewal, differentiation into 1 or more lineages, retention of a DNA synthesis label (BrdU), and some methods to identify them (Hoechst dye exclusion test, immunophenotyping). The most likely markers for endometrial somatic stem cells (Oct-4, Musashi-1, CD31, CD34, and CD144) are also mentioned. The study also reviews the literature regarding endometrial cancer stem cells. Results obtained by evaluations of the side population in endometrial cancer cell lines and studies on putative cancer stem cell markers are also discussed. The possible roles of endometrial cancer stem cells in metastasis and resistance to anticancer treatment are also mentioned.

Annexin-A2 as predictor biomarker of recurrent disease in endometrial cancer

Endometrial carcinomas, the most common malignant tumour of the female genital tract, are usually diagnosed at an early stage with uterine‐confined disease and an overall favourable prognosis. However, up to 20% of endometrial carcinomas will end up in recurrent disease, associated with a drop in survival and representing the major clinical challenge. Management of this group of risk patients relies on robust biomarkers that may predict which endometrial carcinomas will relapse. For this, we performed a proteomic analysis comparing primary lesions with recurrences and identified ANXA2 as a potential biomarker associated with recurrent disease that we further validated in an independent series of samples by immunohistochemistry. We demonstrated in vitro a role for ANXA2 in the promotion of metastasis rather than interfering with sensitivity to radio/chemotherapy. In addition, ANXA2 silencing resulted in a reduced metastatic pattern in a mice model of endometrial cancer dissemination, with a limited presence of circulating tumor cells. Finally, a retrospective study in a cohort of 93 patients showed that ANXA2 effectively predicted those endometrioid endometrial carcinomas that finally recurred. Importantly, ANXA2 demonstrated a predictive value also among low risk Stage I endometrioid endometrial carcinomas, highlighting the clinical utility of ANXA2 biomarker as predictor of recurrent disease in endometrial cancer. Retrospective and prospective studies are ongoing to validate ANXA2 as a potential tool for optimal stratification of patients susceptible to receive radical surgery and radio/chemotherapy.

Combination of Vorinostat and caspase-8 inhibition exhibits high anti-tumoral activity on endometrial cancer cells

Histone deacetylase inhibitors such as Vorinostat display anti‐neoplastic activity against a variety of solid tumors. Here, we have investigated the anti‐tumoral activity of Vorinostat on endometrial cancer cells. We have found that Vorinostat caused cell growth arrest, loss of clonogenic growth and apoptosis of endometrial cancer cells. Vorinostat‐induced the activation of caspase‐8 and ‐9, the initiators caspases of the extrinsic and the intrinsic apoptotic pathways, respectively. Next, we investigated the role of the extrinsic pathway in apoptosis triggered by Vorinostat. We found that Vorinostat caused a dramatic decrease of FLIP mRNA and protein levels. However, overexpression of the long from of FLIP did not block Vorinostat‐induced apoptosis. To further investigate the role of extrinsic apoptotic pathway in Vorinostat‐induced apoptosis, we performed an shRNA‐mediated knock‐down of caspase‐8. Surprisingly, downregulation of caspase‐8 alone caused a marked decrease in clonogenic ability and reduced the growth of endometrial cancer xenografts in vivo, revealing that targeting caspase‐8 may be an attractive target for anticancer therapy on endometrial tumors. Furthermore, combination of caspase‐8 inhibition and Vorinostat treatment caused an enhancement of apoptotic cell death and a further decrease of clonogenic growth of endometrial cancer cells. More importantly, combination of Vorinostat and caspase‐8 inhibition caused a nearly complete inhibition of tumor xenograft growth. Finally, we demonstrate that cell death triggered by Vorinostat alone or in combination with caspase‐8 shRNAs was inhibited by the anti‐apoptotic protein Bcl‐XL. Our results suggest that combinatory therapies using Vorinostat treatment and caspase‐8 inhibition can be an effective treatment for endometrial carcinomas.

ERα-mediated repression of pro-inflammatory cytokine expression by glucocorticoids reveals a crucial role for TNFα and IL1α in lumen formation and maintenance.

Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNFα and IL1α in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNFα and IL1α expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor α (ERα) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ERα-regulated pro-inflammatory cytokine expression in lumen formation and maintenance.

Palbociclib has antitumour effects on Pten‐deficient endometrial neoplasias

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1–CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD‐332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen‐inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten‐deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D–CDK4/6 activity in their development. Copyright