Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

In brief: The regulation of AKT in the endometrium during many cellular processes such as apoptosis and cell survival is crucial during the estrous cycle to ensure fertility. This research shows the specific function of AKT isoforms in the mouse endometrium for litter size, estrous cyclicity and endometrial gland development. Abstract: Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation, and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation is differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in progesterone receptor-expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest not only specific roles between each isoform but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Pharmacologic inhibition of Akt in combination with chemotherapeutic agents effectively induces apoptosis in ovarian and endometrial cancer cell lines.

The PI3K/Akt signaling pathway, the most frequently altered signaling system in human cancer, is a crucial inducer of dysregulated proliferation and neoplastic processes; however, few therapeutic strategies using PI3K/Akt inhibitors singly have been shown to be effective. The purpose of this paper was to underline the potential benefit of pharmacological modulation of the PI3K/Akt pathway when combined with specific chemotherapeutic regimens. We have studied the ability of NVP-BEZ235 (PI3K/mTOR inhibitor) and AZD5363 (Akt inhibitor) in the sensitization of cancer cells to cisplatin and doxorubicin. Our results show that NVP-BEZ235 sensitizes cells preferentially to cisplatin while AZD5363 sensitizes cells to doxorubicin. At equal concentrations (5 µm), both inhibitors reduce ribosomal protein S6 phosphorylation, but AZD5363 is more effective in reducing GSK3ß phosphorylation as well as S6 phosphorylation. Additionally, AZD5363 is capable of inducing FOXO1 and p53 nuclear localization and reduces BAD phosphorylation, which is generally increased by cisplatin and doxorubicin. Finally, the combination of AZD5363 and doxorubicin induces apoptosis in cells and robustly reduces cell ability to clonally replicate, which underlines a potential cooperative effect of the studied compounds.

Inhibition of CRM1 activity sensitizes endometrial and ovarian cell lines to TRAIL-induced cell death.

BACKGROUND: CRM1 enrichment has been shown to be indicative of invasive as well as chemoresistant tumors. On the other hand, TRAIL, a powerful and specific anti-tumoral agent, has yet to be used effectively to treat gynecological tumors in patients. In the present study, we examined if CRM1, a nuclear exporter capable of mediating protein transport, could be a relevant target to restore chemosensitivity in chemoresistant cells. We thus explored the hypothesis that CRM1-driven nuclear exclusion of tumor suppressors could lead to chemoresistance and that CRM1 inhibitors could present a novel therapeutic approach, allowing sensitization to chemotherapeutic agents. METHODS: Ovarian cancer cell lines, as well as endometrial cancer cell lines, were treated with leptomycin B (LMB), cisplatin and TRAIL, either singly or in combination, in order to induce apoptosis. Western blot and flow cytometry analysis were used to quantify caspases activation and apoptosis induction. Immunofluorescence was used to determine nuclear localization of p53. Colony formation assays were performed to determine therapeutic effectiveness; p53 siRNA were used to establish p53 role in sensitization. Additional information from GEO database and Prognoscan allowed us to contextualise the obtained results. Finally, qRT-PCR was performed to measure apoptotic regulators expression. RESULTS: TRAIL and LMB combination therapy lead to cleavage of caspase-3 as well as the appearance of cleaved-PARP, and thus, apoptosis. Further experiments suggested that sensitization was achieved through the synergistic downregulation of multiple inhibitor of apoptosis, as well as the activation of apoptotic pathways. p53 was enriched in the nucleus following LMB treatments, but did not seem to be required for sensitization; additional experiments suggested that p53 opposed the apoptotic effects of LMB and TRAIL. Results obtained from public data repositories suggested that CRM1 was a driver of chemoresistance and poor prognostic; DR5, on the other hand, acted as as a marker of positive prognostic. CONCLUSIONS: Taken together, our results suggest that the use of CRM1 inhibitors, in combination to chemotherapeutic compounds, could be highly effective in the treatment of gynecological malignancies.

PI3K Positively Regulates YAP and TAZ in Mammary Tumorigenesis Through Multiple Signaling Pathways.

Breast cancer is a leading cause of death in women worldwide. Active mutations of PI3K catalytic subunit PIK3CA (e.g., H1047R) and amplification of its homolog PIK3CB are observed in a large number of breast cancers. In recent years, aberrant activation of Transcriptional coactivator with PDZ binding motif (TAZ) and its paralog Yes-associated protein (YAP) have also been found to be important for breast cancer development and progression. However, whether PI3K interacts with YAP/TAZ during mammary tumorigenesis is unknown. Through a systematic gain-of-function screen for kinases involved in mammary tumorigenesis, we identified PIK3CB as a transformation-inducing kinase in breast cells. We further determined that PIK3CB positively regulates YAP and TAZ to promote transformation and inhibit mammary cell death in vitro PIK3CB coexpression with TAZ, rather than PIK3CB or TAZ alone, in human MCF10A nontumorigenic mammary cells is sufficient for tumor formation in mice in vivo Interestingly, we also determined that PIK3CA-H1047R enhances YAP and TAZ activity in mammary tumorigenesis in vitro Mechanistically, the regulation of YAP/TAZ by both PIK3CA and PIK3CB occurs through multiple signaling pathways including LATS-dependent and LATS-independent pathways. Therefore, in this study, we determine that PI3K and YAP/TAZ interact to promote breast cancer cell transformation.Implications: This study provides the first evidence that the Hippo pathway effectors TAZ and YAP are critical mediators of PI3K-induced mammary tumorigenesis and synergistically function together with PI3K in transformation of mammary cells. These findings may provide a novel rationale for targeting YAP/TAZ alone or in combination with PI3K inhibitors for breast cancer therapy in the future. Mol Cancer Res; 16(6); 1046-58. ©2018 AACR.

Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells.

Infertility is constantly increasing in Canada, where 16% of Canadian couples are experiencing difficulty conceiving. It is thought that infertility can emanate from the dysregulated communication between the embryo and the maternal endometrium. In order to allow for this window of implantation to be open at the right moment, endometrial stromal cells proliferate and differentiate by a mechanism called decidualization. Intracellular and molecular mechanisms involved in the regulation of apoptosis and cell proliferation during decidualization of the endometrium are yet to be fully understood. It has been well demonstrated previously that Akt is importantly involved in cell survival and glycogen synthesis. Akt1, Akt2 and Akt3 isoforms have distinct physiological roles; this could also be the case during decidualization and pregnancy. The aim of this study is to investigate the regulation of PI3K/Akt pathway during the decidualization process of endometrial stromal cells. Expression of Akt isoforms, Akt activity (phospho-Akt), pIκB and substrates of Akt during decidualization were measured. To our knowledge, these results are the first to suggest a decrease in levels of Akt isoforms as well as a downregulation of Akt activity in the process of decidualization of human endometrial stromal cells. We also uncovered that decidualization induced nuclear localization of p65 through the phosphorylation of IκB, its inhibitory subunit; however, Par-4, a recently uncovered regulator of cell differentiation, was displaced from the nucleus upon decidualization. Our results also suggest that HIESC cells exhibit decreased motility during decidualization and that PI3K pathway inhibition could be involved in this process. Finally, we demonstrate that specific Akt isoforms present unique effects on the successful induction of decidualization. Further analyses will involve investigations to understand the precise signaling mechanisms by which this pathway is regulated.

Genetic Deletion of Akt3 Induces an Endophenotype Reminiscent of Psychiatric Manifestations in Mice.

The protein kinase B (PKB/Akt), found in three distinctive isoforms (PKBα/Akt1, PKBß/Akt2, PKBγ/Akt3), is implicated in a variety of cellular processes such as cell development, growth and survival. Although Akt3 is the most expressed isoform in the brain, its role in cerebral functions is still unclear. In the present study, we investigated the behavioral, electrophysiological and biochemical consequences of Akt3 deletion in mice. Motor abilities, spatial navigation, recognition memory and LTP are intact in the Akt3 knockout (KO) mice. However, the prepulse inhibition, three-chamber social, forced swim, tail suspension, open field, elevated plus maze and light-dark transition tests revealed an endophenotype reminiscent of psychiatric manifestations such as schizophrenia, anxiety and depression. Biochemical investigations revealed that Akt3 deletion was associated with reduced levels of phosphorylated GSK3α/ß at serine 21/9 in several brain regions, although Akt1 and Akt2 levels were unaffected. Notably, chronic administration of lithium, a mood stabilizer, restored the decreased phosphorylated GSK3α/ß levels and rescued the depressive and anxiety-like behaviors in the Akt3 KO mice. Collectively, our data suggest that Akt3 might be a critical molecule underlying psychiatric-related behaviors in mice.

Chemoresistance and targeted therapies in ovarian and endometrial cancers.

Gynecological cancers are known for being very aggressive at their advanced stages. Indeed, the survival rate of both ovarian and endometrial cancers is very low when diagnosed lately and the success rate of current chemotherapy regimens is not very efficient. One of the main reasons for this low success rate is the acquired chemoresistance of these cancers during their progression. The mechanisms responsible for this acquired chemoresistance are numerous, including efflux pumps, repair mechanisms, survival pathways (PI3K/AKT, MAPK, EGFR, mTOR, estrogen signaling) and tumor suppressors (P53 and Par-4). To overcome these resistances, a new type of therapy has emerged named targeted therapy. The principle of targeted therapy is simple, taking advantage of changes acquired in malignant cancer cells (receptors, proteins, mechanisms) by using compounds specifically targeting these, thus limiting their action on healthy cells. Targeted therapies are emerging and many clinical trials targeting these pathways, frequently involved in chemoresistance, have been tested on gynecological cancers. Despite some targets being less efficient than expected as mono-therapies, the combination of compounds seems to be the promising avenue. For instance, we demonstrate using ChIP-seq analysis that estrogen downregulate tumor suppressor Par-4 in hormone-dependent cells by directly binding to its DNA regulatory elements and inhibiting estrogen signaling could reinstate Par-4 apoptosis-inducing abilities. This review will focus on the chemoresistance mechanisms and the clinical trials of targeted therapies associated with these, specifically for endometrial and ovarian cancers.

Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells.

We recently reported the caspase3-dependent cleavage of Par-4 resulting in the accumulation of a 25kDa cleaved-Par-4 (cl-Par-4) fragment and we investigated in the present study the mechanisms regulating this fragment using cl-Par-4-expressing stable clones derived from ovarian and endometrial cancer cell lines.Cl-Par-4 protein was weakly express in all stable clones despite constitutive expression. However, upon cisplatin treatment, cl-Par-4 levels increased up to 50-fold relative to baseline conditions. Treatment of stable clones with proteasome and translation inhibitors revealed that cisplatin exposure might in fact protect cl-Par-4 from proteasome-dependent degradation. PI3K and MAPK pathways were also implicated as evidenced by an increase of cl-Par-4 in the presence of PI3K inhibitors and a decrease using MAPK inhibitors. Finally using bioinformatics resources, we found diverse datasets showing similar results to those we observed with the proteasome and cl-Par-4 further supporting our data.These new findings add to the complex mechanisms regulating Par-4 expression and activity, and justify further studies addressing the biological significance of this phenomenon in gynaecological cancer cells.

Parasporin-2 from a New Bacillus thuringiensis 4R2 Strain Induces Caspases Activation and Apoptosis in Human Cancer Cells.

In previous studies, parasporin-2Aa1, originally isolated from Bacillus thuringiensis strain A1547, was shown to be cytotoxic against specific human cancer cells but the mechanisms of action were not studied. In the present study, we found that proteinase K activated parasporin-2Aa1 protein isolated from a novel B. thuringiensis strain, 4R2, was specifically cytotoxic to endometrial, colon, liver, cervix, breast and prostate cancer. It showed no toxicity against normal cells. Upon treatment with proteinase K-activated parasporin-2Aa1, morphological changes were observed and western blot analysis revealed the cleavage of poly (ADP-Ribose) polymerase, caspase-3 and caspase-9 in cancer cell lines exclusively, indicative of programmed cell death, apoptosis. Flow cytometry analyses,using propidium iodide and annexin V, as well as a caspases 3/7 assay confirmed apoptosis induction. Further analyses were performed to study survival pathways, including AKT, XIAP, ERK1/2 and PAR-4, a known inducer of apoptosis. These results indicate that parasporin-2Aa1 is a selective cytotoxic protein that induces apoptosis in various human cancer cell lines from diverse tissues.

Exploring the synthesis and anticancer potential of L-tyrosine-platinum(II) hybrid molecules.

The search for new specific chemotherapeutic drugs designed to minimize the toxic side effects resulting from chemotherapy is still a subject of intense research. The objective of the current study was to design a non-steroidal-platinum(II) derivative that would target the estrogen receptor alpha (ERα) without triggering estrogenic cell proliferation. For this purpose, the amino acid L-tyrosine was modified and attached to a cisplatin analog. Hence, the L-tyrosine portion of the molecule could possibly act as a transporter to target the ERα protein and, by doing so concentrate the cytotoxic moiety to hormone-dependent breast cancer cells. Herein, we describe three different alternative methodologies that were used to make these new anticancer molecules. The L-tyrosine-Pt(II) hybrid 5b was made in four steps with 36% overall yield by the first method, in six steps with 11% overall yield by the second method and, in four steps with 23% overall yield by the third method. Preliminary biological activity on breast cancer cell lines indicated that the final hybrids (5a and 5b) were unfortunately inactive but their platinum(II) precursors (14a and 14b) showed activity similar to that of cisplatin.

New testosterone derivatives as semi-synthetic anticancer agents against prostate cancer: synthesis and preliminary biological evaluation.

Prostate cancer (PC) is a major health issue in the world. Treatments of localized PC are quite efficient and usually involve surgery, radiotherapy and/or hormonal therapy. Metastatic PC is however rarely curable to this day. Treatments of metastatic PC involve radiotherapy, chemotherapy and hormonal treatment such as orchiectomy, antiandrogens and luteinizing hormone-releasing hormone agonists. The suppression of tumor growth by hormonal treatment is efficient but overtime resistance still occurs and the disease progresses. Thus, more urgently than ever there is a need for discovery of new treatment options for castration-resistant PC (CRPC). Hence, we designed and tested a series of amide derivatives located at position 7α of testosterone as prospective "natural" or "semisynthetic" anticancer agents against CRPC with the goal of discovering therapeutic alternatives for the disease. This manuscript describes an efficient path towards the target molecules that are made in only 6 or 7 chemical steps from testosterone in good overall yields. This strategy can be used to make several compounds of interest that present higher biological activity than the classic antiandrogen; cyproterone acetate (3). The best testosterone-7α-amide was the N-2-pyridylethylamide (25) which was as active as the antiandrogen cyproterone acetate (3) on androgen-dependent LNCaP cells and 2.7 times more active on androgen-independent PC3 prostate cancer cells. The results obtained show the synthetic feasibility and the potential for future development of this unique class of semi-synthetic anticancer agents that offer the premise of new treatment modalities for patients afflicted with CRPC.

Expression, activation, and role of AKT isoforms in the uterus.

The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases.

Resveratrol inhibits cisplatin-induced epithelial-to-mesenchymal transition in ovarian cancer cell lines.

BACKGROUND: Many patients diagnosed with ovarian cancer experience recurrence and metastasis, two aspects that will often cause their demise. Epithelial-to-mesenchymal transition (EMT) is a key process involved in cancer progression. With increasing evidence linking Cisplatin and EMT, we wanted to identify a compound able to counter EMT progression when cancer cells are treated with Cisplatin. METHODOLOGY/PRINCIPAL FINDINGS: Cell death was evaluated by cytometry with Annexin V/PI staining in A2780 and A2780CP cells. Ovarian cancer cell lines were treated with Cisplatin (24 h, 10 µM) and different concentrations of Resveratrol to evaluate its effect on Cisplatin-induced EMT using Western Blot and RT-PCR analysis. Morphological studies and wound healing assay to evaluate cell motility were performed using 72 h Cisplatin treatment with A2780 and A2780CP cells. Densitometry was done on Western Blot and PCR results, and statistical significance was determined using One-Way ANOVA followed by Tukey post-hoc test. Our results show that Cisplatin induced EMT-associated morphological changes in the A2780 ovarian cancer cell line and to a lesser extent in its Cisplatin-resistant counterpart A2780CP. Resveratrol caused cell death in A2780 and A2780CP cell lines in an apoptotic-independent manner. Resveratrol inhibited Cisplatin-induced Snail expression by reducing the Erk pathway activation, reverted morphological changes induced by Cisplatin and decreased cell migration. CONCLUSIONS: These results indicate that Resveratrol has interesting potential to prevent Cisplatin-induced EMT in ovarian cancer cells. By increasing cell death, it also represents an inviting approach as adjuvant therapy to be used with chemotherapy. Using Erk pathway inhibitors could also prove helpful in ovarian cancer treatment to reduce the risk of metastasis.

New platinum(II) complexes conjugated at position 7α of 17ß-acetyl-testosterone as new combi-molecules against prostate cancer: design, synthesis, structure-activity relationships and biological evaluation.

Prostate cancer is a major public health problem worldwide and, more specifically, new treatments for hormone-refractory cancers are highly sought by several research groups. Although platinum(II)-based chemotherapy and other strategies grow in interest to treat castration-resistant prostate cancer (CRPC), they still exhibit modest activity on CRPC and overall patient survival. In this study, we designed and prepared new combi-molecules using 17ß-acetyl-testosterone and amino acid platinum(II) complexes linked at the position 7α to target and to improve the antiproliferative activity of platinum(II)-based chemotherapy on prostate cancer cells. Twelve chemical intermediates and six new combi-molecules were prepared and characterized. Structure-activity relationships studies show that the platinum complex moiety is essential for an optimal cytocidal activity. Moreover, stereochemistry of the amino acid involved in the platinum complexes had only minor effects on the antiproliferative activity whereas pyridinyl (10a and b) and thiazolyl (10f) complexes exhibited the highest cytocidal activities that are significantly superior to that of cisplatin used as control on human prostate adenocarcinoma LNCaP (AR+), PC3 (AR-) and DU145 (AR-). Compounds 10a, b and f arrested the cell cycle progression in S-phase and induced double strand breaks as confirmed by the phosphorylation of histone H2AX into γH2AX. Compounds 10a and f showed 33 and 30% inhibition, respectively of the growth of HT-1080 tumors grafted onto chick chorioallantoic membranes. Finally, compounds 10a and 10f exhibited low toxicity on the chick embryos (18 and 21% of death, respectively), indicating that these new combi-molecules might be a promising new class of anticancer agents for prostate cancer.

Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.

BACKGROUND: The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. METHODS: Cells were treated with 10µM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. RESULTS: PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. CONCLUSION: The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

Synthesis and preliminary in vitro biological evaluation of 7α-testosterone-chlorambucil hybrid designed for the treatment of prostate cancer.

The synthesis of 7α-testosterone-chlorambucil hybrid is reported. This compound is made from testosterone in a 6 step reaction sequence and with 23% overall yield. An alternative convergent reaction sequence yielded the same hybrid through a Grubbs metathesis reaction between chlorambucil allyl ester and 7α-allyltestosterone with 35% overall yield. MTT assays showed that the hybrid is selective towards hormone-dependent prostate cancer cell line (LNCaP (AR+)) and shows similar activity than the parent drug, chlorambucil. Thus, the new hybrid shows promising potential for drug targeting of hormone-dependent prostate cancer through its capacity of delivering chlorambucil directly to the site of treatment. This could extend the use of chlorambucil to prostate cancer in the future.

Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation.

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Regulation of the PI3-K/Akt survival pathway in the rat endometrium.

The occurrence of apoptosis and cell survival in the receptive uterus is intimately involved in the embryo implantation process in order to facilitate embryo attachment to the maternal endometrium. The initial stimulus leading to successful implantation might be triggered by the conceptus itself. By the end of rat embryo implantation, decidualization begins, followed by the regression of the decidua basalis on Day 14. The phosphatidylinositol 3-kinase (PI3-K) survival pathway and TGF-beta have been thought to play a role in this process. The objective of the present study was to investigate the regulation of the PI3-K/PTEN/Akt pathway in rat endometrium during pregnancy. Rats were killed on different days of pregnancy (Day 1-22 and postpartum) or pseudopregnancy (Day 1-9), and uteri were removed to collect endometrial tissues. The active form of Akt (pAkt) was increased at Day 5 of pregnancy and at Day 3 of pseudopregnancy as well as at Day 12 of pregnancy and at Day 1 postpartum. Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt3 was the only isoform phosphorylated at Day 5 during the implantation process and at postpartum as demonstrated by immunoprecipitation studies. PI3-K inhibition in vivo blocked Akt phosphorylation, reduced Smad2 phosphorylation, and reduced both TGF-beta2 and XIAP expression. PI3-K inhibition in cultured decidual cells led to inhibition of pAkt and decrease XIAP expression. These results suggest that Akt and XIAP may be important surviving signaling molecules by which apoptosis is regulated in the rat endometrium during pregnancy and that TGF-beta could be linked to this process.

CD40 pathway activation reveals dual function for macrophages in human endometrial cancer cell survival and invasion.

Reproductive malignancies are a major cause of cancer death in women worldwide. CD40 is a TNF receptor family member, which upon activation may mediate tumor regression. However, despite the great potential of CD40 agonists, their use as a therapeutic option for reproductive cancers has never been investigated. Because CD40 ligation is a potent pathway of macrophage activation, an in vitro model of pro-inflammatory type-1 (Mϕ-1) and anti-inflammatory type-2 (Mϕ-2) macrophages was developed to determine whether and how macrophage CD40 pathway activation might influence endometrial tumor cell behavior. Analysis of tumor growth kinetic in the endometrial cancer xenograft model indicates that, when injected once into the growing tumors, CD40-activated Mϕ-1 greatly reduced, while CD40-activated Mϕ-2 increased tumor size when compared to control isotype-activated Mϕ-1 and Mϕ-2, respectively. In vitro assays indicated that CD40-activated Mϕ-2 increased cell viability but failed to promote cell invasion. CD40-activated Mϕ-1, in contrast, decreased cell survival but greatly increased cell invasion in tumor cells less susceptible to cell death by apoptosis; they also induced the expression of some pro-inflammatory genes, such as IL-6, LIF, and TNF-α, known to be involved in tumor promotion and metastasis. The presence of IFN-γ is minimally required for CD40-activated Mϕ-1 to promote tumor cell invasion, a process that is mediated in part through the activation of the PI3K/Akt2 signaling pathway in tumor cells. From these results, we speculate that some functions of CD40 in tumor-associated Mϕs might limit the therapeutic development of CD40 agonists in endometrial cancer malignancies.

Involvement of Akt isoforms in chemoresistance of endometrial carcinoma cells.

OBJECTIVE: In tumors, upstream regulation of Akt is affected by oncogenic events which lead to its constitutive activation and promote cell survival. Since studies have demonstrated that the three Akt isoforms exhibit different physiological functions, Akt isoforms may contribute differently in chemoresistance. The objective of the study was to determine the role of each Akt isoforms in chemoresistance. METHODS: We stably transfected the chemoresistant KLE endometrial carcinoma cells with specific shRNAs for Akt1, Akt2 or Akt3. Alternatively, we stably transfected the chemosensitive Hec-1-A endometrial carcinoma cells, in which no Akt activity is detected, with constitutively active Akt expression vectors for each isoform. RESULTS: We demonstrated that Akt1 and Akt2 downregulation by RNAi highly sensitizes KLE cells to cisplatin by inducing the activation of pro-apoptotic factors such as the cleavage of caspases-3, -6, -9 and PARP; downregulation of all Akt isoforms leads to increased sensitivity to doxorubicin while only Akt1-2 downregulation increases taxol sensitivity. Proliferation of Akt1, and mostly Akt2 deficient cells was affected by cisplatin treatment. Constitutive Akt1 or Akt2 expression led to an increased resistance to apoptosis. Akt isoforms have been shown to influence migration in other cancer cells. We showed that Akt2 blocks cell motility, while Akt1-3 had less effect on our endometrial cancer cell models. CONCLUSION: Our findings highlight the contribution of Akt1 and Akt2 in the molecular mechanisms that govern chemoresistance of endometrial carcinomas. Furthermore, Akt isoform-specific transfectants will provide a strong model to determine the involvement of each Akt isoform in tumor progression and metastasis.