Henrique J. Cardoso

Estrogens down-regulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effects.

The development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.

Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility?

The c-KIT, a tyrosine kinase receptor, and its ligand the stem cell factor (SCF) play an important role in the production of male and female gametes. The interaction of SCF with c-KIT is required for germ cell survival and growth, and abnormalities in the activity of the SCF/c-KIT system have been associated with human infertility. Recently, it was demonstrated that gonadotropic and sex steroid hormones, among others, regulate the expression of SCF and c-KIT in testicular and ovarian cells. Therefore, the hormonal (de)regulation of SCF/c-KIT system in the testis and ovary may be a cause underpinning infertility. In the present review, we will discuss the effects of hormones modulating the expression levels of SCF and c-KIT in the human gonads. In addition, the implications of hormonal regulation of SCF/c-KIT system for germ cell development and fertility will be highlighted.

The SCF/c-KIT system in the male: Survival strategies in fertility and cancer

Maintaining the delicate balance between cell survival and death is of the utmost importance for the proper development of germ cells and subsequent fertility. On the other hand, the fine regulation of tissue homeostasis by mechanisms that control cell fate is a factor that can prevent carcinogenesis. c‐KIT is a type III receptor tyrosine kinase activated by its ligand, stem cell factor (SCF). c‐KIT signaling plays a crucial role in cell fate decisions, specifically controlling cell proliferation, differentiation, survival, and apoptosis. Indeed, deregulating the SCF/c‐KIT system by attenuation or overactivation of its signaling strength is linked to male infertility and cancer, and rebalancing its activity via c‐KIT inhibitors has proven beneficial in treating human tumors that contain gain‐of‐function mutations or overexpress c‐KIT. This review addresses the roles of SCF and c‐KIT in the male reproductive tract, and discusses the potential application of c‐KIT target therapies in disorders of the reproductive system. Mol. Reprod. Dev. 81: 1064–1079, 2014.

Paradoxical and Contradictory Effects of Imatinib in Two Cell Line Models of Hormone-Refractory Prostate Cancer

Imatinib mesylate is a chemotherapeutic drug that inhibits the tyrosine kinase activity of c‐KIT and has been successfully used to treat leukemias and some solid tumors. However, its application for treatment of hormone‐refractory prostate cancer (HRPC) has shown modest effectiveness and did not follow the outcomes in cultured cells or animal models. Moreover, the molecular pathways by which imatinib induces cytotoxicity in prostate cancer cells are poorly characterized.

The stem cell factor (SCF)/c-KIT signalling in testis and prostate cancer

The stem cell factor (SCF) is a cytokine that specifically binds the tyrosine kinase receptor c-KIT. The SCF/c-KIT interaction leads to receptor dimerization, activation of kinase activity and initiation of several signal transduction pathways that control cell proliferation, apoptosis, differentiation and migration in several tissues. The activity of SCF/c-KIT system is linked with the phosphatidylinositol 3-kinase (PI3-K), the Src, the Janus kinase/signal transducers and activators of transcription (JAK/STAT), the phospholipase-C (PLC-γ) and the mitogen-activated protein kinase (MAPK) pathways. Moreover, it has been reported that cancer cases display an overactivation of c-KIT due to the presence of gain-of-function mutations or receptor overexpression, which renders c-KIT a tempting target for cancer treatment. In the case of male cancers the most documented activated pathways are the PI3-K and Src, both enhancing abnormal cell proliferation. It is also known that the Src activity in prostate cancer cases depends on the presence of tr-KIT, the cytoplasmic truncated variant of c-KIT that is specifically expressed in tumour tissues and, thus, a very interesting target for drug development. The present review provides an overview of the signalling pathways activated by SCF/c-KIT and discusses the potential application of c-KIT inhibitors for treatment of testicular and prostatic cancers.

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers.

Suppressed glycolytic metabolism in the prostate of transgenic rats overexpressing calcium-binding protein regucalcin underpins reduced cell proliferation

Regucalcin (RGN) is a calcium-binding protein underexpressed in human prostate cancer cases, and it has been associated with the suppression of cell proliferation and the regulation of several metabolic pathways. On the other hand, it is known that the metabolic reprogramming with augmented glycolytic metabolism and enhanced proliferative capability is a characteristic of prostate cancer cells. The present study investigated the influence of RGN on the glycolytic metabolism of rat prostate by comparing transgenic adult animals overexpressing RGN (Tg-RGN) with their wild-type counterparts. Glucose consumption was significantly decreased in the prostate of Tg-RGN animals relatively to wild-type, and accompanied by the diminished expression of glucose transporter 3 and glycolytic enzyme phosphofructokinase. Also, prostates of Tg-RGN animals displayed lower lactate levels, which resulted from the diminished expression/activity of lactate dehydrogenase. The expression of the monocarboxylate transporter 4 responsible for the export of lactate to the extracellular space was also diminished with RGN overexpression. These results showed the effect of RGN in inhibiting the glycolytic metabolism in rat prostate, which was underpinned by a reduced cell proliferation index. The present findings also suggest that the loss of RGN may predispose to a hyper glycolytic profile and fostered proliferation of prostate cells.

The Emerging Role of Regucalcin as a Tumor Suppressor: Facts and Views

Regucalcin (RGN) is a multifunctional protein that was first described as a calcium (Ca2+)-binding protein playing a relevant role in the maintenance of intracellular Ca2+ concentration. However, due to its downregulated expression with aging, RGN is also known as senescence marker protein-30. The RGN protein is an X-chromosome gene product, whose transcription is regulated by a myriad of hormonal and non-hormonal factors. Besides the well-known role in Ca2+ homeostasis, RGN has also been linked to the control of several intracellular signaling pathways, and basic biological processes, such as oxidative stress, cell proliferation, apoptosis, and metabolism. RGN has been shown to have antioxidant properties by its activity reducing the production of reactive oxygen species and increasing the antioxidant defenses. The role of RGN suppressing cell proliferation is associated with the regulation of expression of oncogenes and tumor suppressor genes. It results clear that all the existent knowledge implicates RGN in the control of the main biological processes actually recognized as the hallmarks of cancer. Moreover, it has been shown that tumor onset and progression are underpinned by the loss of RGN expression, whereas RGN overexpression showed to have a protective role against the development of chemicallyinduced tumors. This review describes the mechanisms that control the tissue expression of RGN and discusses the experimental evidence that indicate RGN as a new tumor suppressor protein.

The Role of GPER Signaling in Carcinogenesis: A Focus on Prostate Cancer

The G protein-coupled estrogen receptor, GPER, also known as GPR30, belongs to the seven transmembrane receptor superfamily and is involved in the rapid non-genomic estrogenic responses. Nevertheless, GPER regulation of transcriptional activity also has been reported. GPER downstream signaling includes the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, the stimulation of adenylyl cyclase, and the mobilization of intracellular calcium. Over the last decade, the discovery of GPER specific agonists and antagonists has been crucial to understand its physiological functions, mechanisms of action and its putative usefulness as a therapeutic target. The GPER seems to have an important role in endocrine, reproductive, immune, nervous, and cardiovascular systems, and alterations in its expression or activity have been associated with several pathological conditions such as cardiovascular diseases, obesity, diabetes, Parkinson, stroke, and cancer. GPER has been linked with the carcinogenic process, though some ambiguity exists concerning its protective or causative role in different tissues, or even in the same tissue. This chapter summarizes the existent knowledge concerning the structural and molecular aspects of GPER, its known ligands and activated pathways, as well as its role over the known hallmarks of cancer: exacerbated proliferation, resistance to apoptosis, stimulated migration and invasion, induction of angiogenesis, and the metabolic reprogramming. A special focus will be given to prostate cancer.

PO-255 The pivotal role of glutaminolysis in prostate cancer cells and its regulation by androgens

Introduction Progression of prostate cancer (PCa) is initially dependent on androgens enabling the use of therapies reducing androgens production or antagonising the androgen receptor (AR). However, the majority of cases progress to a castrate-resistant stage (CRPC) characterised by the proliferation of PCa cells independently of androgen depletion. Previous work showed that progression of PCa to CRPC is associated with a metabolic adaptation. Also, we demonstrated that androgens modulate the glycolytic metabolism in PCa cells. However, the effect of androgens in glutaminolysis is unknown. Herein, we characterised the expression of key players in glutamine metabolism; investigated the effect of androgens in glutaminolysis; and evaluated the effect of glutaminase inhibition in PCa cells. Material and methods Western Blot analysis characterised the expression of the glutamine transporter, ASCT2, and glutaminase in LNCaP, DU145 and PC3 cells. PCa cells and male rats were exposed to 10 nM and 500 mg/kg/day of 5a-dihydrotestosterone (DHT), respectively, and the expression of ASCT2 and glutaminase was assessed. Viability of PCa cells was evaluated by the MTT assay in the absence or presence of glutamine and DHT, as well as after inhibiting glutaminase and AR. The effect of glutaminase inhibition was evaluated by the MTT and migration assays, caspase-3 activity and expression P53, p21 and c-myc. The effect of glutaminase inhibition on glycolytic metabolism was also evaluated. Results and discussions DU145 and PC3 cells displayed decreased expression of ASCT2 and increased expression of glutaminase relatively to LNCaP. DHT treatment modulated the expression of ASCT2 and glutaminase in LNCaP cells and rat prostate. The presence of glutamine in culture medium promoted viability of CRPC cells in contrast with the observed in LNCaP. BPTES, a specific inhibitor of glutaminase, diminished cell viability, decreased migration and c-myc expression, and increased caspase-3 activity and p21 expression. Inhibition of glutaminolysis with BPTES, also affected the glycolytic metabolism decreasing the expression of several transporters and enzymes. Moreover, BPTES in co-treatment with bicalutamide, an AR inhibitor, was more effective suppressing cell viability and migration. Conclusion These findings showed that the expression of glutaminolysis associated-proteins is dependent on androgens and PCa aggressiveness, and that the co-inhibition of AR and glutaminase should be investigated as a new therapy.