Browsing by Subject "PI3K"

Now showing 1 - 7 of 7

Open Access
Cholesterol biogenesis is a PTEN-dependent actionable node for the treatment of endocrine therapy-refractory cancers
(Wiley, 2023-09-14) Kaysudu, Irmak; Güngül, T. B.; Atıcı, S.; Yılmaz, S.; Bayram, E.; Güven, G.; Çizmecioğlu, N. T.; Şahin, Ö.; Yeşilöz, Gürkan; Haznedaroğlu, B. Z.; Çizmecioğlu, Onur
PTEN and PIK3CA mutations are the most prevalent PI3K pathway alterations in prostate, breast, colorectal, and endometrial cancers. p110β becomes the prominent PI3K isoform upon PTEN loss. In this study, we aimed to understand the molecular mechanisms of PI3K dependence in the absence of PTEN. Using online bioinformatical tools, we examined two publicly available microarray datasets with aberrant PI3K activation. We found that the rate-limiting enzyme of cholesterol biogenesis, SQLE, was significantly upregulated in p110β-hyperactivated or PTEN-deficient mouse prostate tumors. Concomitantly, the expression of cholesterol biosynthesis pathway enzymes was directly correlated with PI3K activation status in microarray datasets and diminished upon PTEN re-expression in PTEN-null prostate cancer cells. Particularly, PTEN re-expression decreased SQLE protein levels in PTEN-deficient prostate cancer cells. We performed targeted metabolomics and detected reduced levels of cholesteryl esters as well as free cholesterol upon PTEN re-expression. Notably, PTEN-null prostate and breast cancer cell lines were more sensitive to pharmacological intervention with the cholesterol pathway than PTEN-replete cancer cells. Since steroid hormones use sterols as structural precursors, we studied whether cholesterol biosynthesis may be a metabolic vulnerability that enhances antihormone therapy in PTEN-null castration-resistant prostate cancer cells. Coinhibition of cholesterol biosynthesis and the androgen receptor enhanced their sensitivity. Moreover, PTEN suppression in endocrine therapy-resistant luminal-A breast cancer cells leads to an increase in SQLE expression and a corresponding sensitization to the inhibition of cholesterol synthesis. According to our data, targeting cholesterol biosynthesis in combination with the hormone receptor signaling axis can potentially treat hormone-resistant prostate and breast cancers.
Open Access
Identification and targeting of deregulated metabolic pathways in metastatic prostate cancer cells
(2023-01) Kaysudu, Irmak
Prostate cancer is the most diagnosed cancer type and the second leading cause of death in men globally. The pathogenesis of prostate cancer mainly relies on the androgen signaling axis. Therefore, androgen deprivation therapy is the primary treatment for prostate cancer. Nevertheless, the disease progression proceeds, followed by castration resistance and androgen independence. Aberrant androgen signaling activity intertwined with the hyperactivated PI3K-Akt signaling pathway has important oncogenic consequences for castration resistance mechanisms. PTEN, a negative regulator of the PI3K/Akt pathway, is one of the most altered tumor suppressor genes in prostate cancer. PTEN loss occurs in the initial stages of prostate cancer and the frequency of its alteration increases in metastatic and castration-resistant prostate cancer. PTEN has both lipid and protein phosphatase activity, with the former antagonizing the PI3K-Akt pathway by converting membrane-associated PIP3 to PIP2. PTEN loss may cause metabolic rewiring in metastatic prostate cancer cells and the associated metabolic vulnerabilities may be tackled for the disease therapy. To understand the impact of PTEN loss in metastatic prostate cancer cells, we created a dox-inducible system in PTEN-null metastatic and castration-naïve LNCaP cells to re-express WT-PTEN and various PTEN functional mutants, and we employed targeted metastatic prostate cancer. Our multidirectional omics studies suggest that the acquisition of resistance to castration depends on the deregulation of the sphingolipid metabolism in metastatic prostate cancer cells. Furthermore, we showed that PTEN re-expression in metastatic and castration-naïve LNCaP cells attenuated sphingosine kinase levels, which might switch the sphingolipid metabolism towards increased sphingomyelin biosynthesis and ceramide phosphorylation. Moreover, we showed decreased PI3K/Akt pathway activity when we inhibited sphingosine kinase with opaganib in LNCaP cells. Our results also showed a significant upregulation in sphingolipid metabolism in castration-resistant C4-2 cells compared to castration-naïve LNCaP. We treated these cells with several sphingolipid metabolism inhibitors and discovered that castration-resistant prostate cancer cells were more sensitive to opaganib or ARN14988, but not to fingolimod, than castration-naïve prostate cancer cells. These findings suggest that sphingolipid metabolism might be a promising target for the treatment of metastatic and castration-resistant prostate cancer. Understanding changes in sphingolipid metabolism may be critical for developing rational combinatorial targeting strategies for prostate cancer in the long run.
Open Access
Inhibition of Akt signaling in hepatoma cells induces apoptotic cell death independent of Akt activation status
(Springer, 2011) Buontempo, F.; Ersahin, T.; Missiroli, S.; Senturk, S.; Etro, D.; Ozturk, M.; Capitani, S.; Cetin Atalay, R.; Neri, M. L.
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is involved in cell survival and anti-apoptotic signaling. Akt has been shown to be constitutively expressed in a variety of human tumors including hepatocellular carcinoma (HCC). In this report we analyzed the status of Akt pathway in three HCC cell lines, and tested cytotoxic effects of Akt pathway inhibitors LY294002, Wortmannin and Inhibitor VIII. In Mahlavu human hepatoma cells Akt was constitutively activated, as demonstrated by its Ser473 phosphorylation, downstream hyperphosphorylation of BAD on Ser136, and by a specific cell-free kinase assay. In contrast, Huh7 and HepG2 did not show hyperactivation when tested by the same criteria. Akt enzyme hyperactivation in Mahlavu was associated with a loss of PTEN protein expression. Akt signaling was inhibited by the upstream kinase inhibitors, LY294002, Wortmannin, as well as by the specific Akt Inhibitor VIII in all three hepatoma cell lines. Cytotoxicity assays with Akt inhibitors in the same cell lines indicated that they were all sensitive, but with different IC50 values as assayed by RT-CES. We also demonstrated that the cytotoxic effect was through apoptotic cell death. Our findings provide evidence for its constitutive activation in one HCC cell line, and that HCC cell lines, independent of their Akt activation status respond to Akt inhibitors by apoptotic cell death. Thus, Akt inhibition may be considered as an attractive therapeutic intervention in liver cancer. © Springer Science+Business Media, LLC 2010.
Open Access
Investigation of PI3K functional compensation VIA activated tyrosine kinases
(2020-12) Demir, Melike
Protein tyrosine kinases and serine-threonine kinases have crucial functions in cell signaling, differentiation, motility, and proliferation. PI3K is the most deregulated pathway in human cancers and an essential regulator of cellular proliferation. PI3K pathway is activated via oncogenic Ras/receptor tyrosine kinases (RTKs), PTEN loss, or activating mutations in PI3Ks. Moreover, PI3K is one of the most promising pathways for targeted therapies. Thus, many serine-threonine or tyrosine kinases contribute to drug resistance elicited by PI3K inhibition. In order to identify an individual tyrosine kinase that contributes to PI3K functional compensation, the activated tyrosine kinase library was screened and found out that ZAP70 can compensate growth upon PI3K abrogation. This study suggests a mechanism of activated ZAP70 mediated partial resistance in MEFs lines. Moreover, we demonstrated the role of activated tyrosine kinase, ZAP70, in cancer cells as a tumorinitiating factor. Activated ZAP70 is able to enhance the growth ability of untransformed cells. Also in these cells, activated ZAP70 can develop partial resistance to PI3K inhibition. This resistance occurs via activated downstream targets of tyrosine kinase signaling such as STAT3/MAPK axis. Furthermore, we showed that activated ZAP70 has a high transformation capability associated with the formation of malignant phenotype in untransformed cells. Overall, ZAP70 may be a potent driver of proliferation and transformation in untransformed cells as well as in cancer cells.
Open Access
Molecular mechanisms of PI3K isoform dependence in carcinogenesis
(2020-12) Atıcı, Sena
PI3K pathway is important for cellular proliferation, survival and metabolism. PTENloss and activating mutations of PIK3CA are most frequently seen PI3K related alterations in various cancer types. Activating mutations in PIK3CA could render tumors p110α dependent. Also, upon PTEN-loss, p110β isoform of ClassIA PI3Ks becomes prominent. Nevertheless, the mode of action of p110 prevalence is still not clear. Here, we aimed to understand the mechanism of the isoform dependence switch in PTEN-null cancer types. Firstly, we found that PTEN status had an impact on isoform prevalence in both MEFs and PC3s. p110α dependence decreased in PTENdepleted MEFs and reciprocally there was a decrease in dependence to p110β in PTEN-reexpressed PC3 cells. On the other hand, upon modulation of PTEN expression, there was no complete switch-over in dependence from one ClassIA PI3K isoform to the other. Interestingly, when p110β overexpression was performed in PTEN depleted MEFs, cells became less dependent on p110α and more dependent on p110β for cellular viability. However, p110β overexpression in combination with PTEN status change did not again induce a complete isoform switch within ClassIA PI3Ks. To reveal additional modules involved in PI3K isoform prevalence, GSE21543 dataset was analyzed and mRNA levels of AK4, SQLE, CREB3L4 genes were found to be significantly upregulated in constitutively activated p110β in murine models of prostate cancer. Among these genes, SQLE,a rate limiting enzyme in cholesterol synthesis, is highly amplified in various tumor samples according to patient datasets. Breast and prostate cancers have relatively higher amplification rates of SQLE compared to other cancer types. The simultaneous incidence of PTEN mutation and SQLE amplification rate is also frequently observed in breast and prostate cancers. mRNA levels of cholesterol synthesis genes were upregulated in GSE21543 dataset when the PI3K pathway was constitutively activated by p110β myristoylation. Also, the expression levels of the cholesterol synthesis genes were decreased upon PI3K repression with PTEN re-expression in our qPCR results. Constitutively activated p110β MEFs and PTEN-null prostate, breast cancer cell lines were found to be sensitive to inhibitors of rate-limiting enzymes of cholesterol synthesis pathway. Besides in-line with mRNA levels, SQLE protein levels were decreased in PTEN reexpressed prostate cancer cell lines. On the other hand, SQLE protein levels were stabilized upon PTEN expression with protease inhibitor treatment which indicates that PTEN and PI3K activity may affect SQLE transcriptionally as well as posttranslationally. We also demonstrated that tamoxifen therapy responders have lower survival rate and higher SQLE expression according to patient data. In line with this data, in cellular models of tamoxifen resistant breast cancer, we have seen elevated levels of SQLE. All in all, our data emphasizes the critical importance of cholesterol synthesis pathway as a metabolic effector of the PI3K pathway and we can speculate that p110β dependence in PTEN-null cancer types might arise as a result of its excessive activation.
Open Access
Systems biology approach for targeted therapy of liver cancer P13K/AKT/MTOR pathway inhibitors : an ally or rival for sorafenib'e
(2014) Erşahin, Tülin
Hepatocellular carcinoma (HCC) is one of the leading causes of cancerrelated mortality worldwide. It is the second most frequent cause of cancer death in men, and the sixth in women due to its aggressive behavior and resistance to conventional therapies. Sorafenib (Nexavar, BAY43-9006), a multi-kinase inhibitor with anti-angiogenic functions, is the only FDA-approved molecular-targeted agent for the treatment of patients with advanced HCC. Yet, Sorafenib shows limited overall survival benefit associated with resistance and tumor recurrence. Current mono-target- or single pathway-centric drug designs are not sufficient for effective therapy of advanced HCC. Negative results of clinical trials on targeted therapies for advanced HCC are due to clinical heterogeneity, complexity of cirrhotic background and interconnected regulation of cancer hallmarks through compensatory signaling pathways with redundant functions. Secretion of growth factors, pro-inflammatory and immune-suppressive cytokines and chemokines in the tumor microenvironment and consequent activation of tumor-promoting signaling cascades confer resistance to Sorafenib treatment. RAF/MEK/ERK and PI3K/AKT/mTOR are the major tumorpromoting signaling pathways with regulatory functions in all hallmarks of HCC. They have redundant functions and inhibition of one pathway can stimulate compensatory signaling from the other pathway. Since Sorafenib targets angiogenic VEGFR and PDGFR kinases and RAF/MEK/ERK signaling, the primary mechanism of resistance to Sorafenib and tumor recurrence in HCC patients emerges to be the compensatory signaling from the PI3K/AKT pathway. Therefore, we anticipated that combined treatment with Sorafenib and PI3K/AKT inhibitors could reverse drug resistance in HCC. In this study, we analyzed the synergistic effects of Sorafenib and PI3K/AKT inhibitors on HCC cell growth and migration, determined possible mechanisms underlying synergistic mechanism of action by transcriptome analysis, and further showed that combination therapy leads to tumor regression in PTEN-deficient HCC xenografts in vivo. We showed that PTEN-deficient HCC cells with constitutively active PI3K/AKT signaling depend on the alpha isoform of PI3K (p110-α) for survival and co-targeting these cells with the isoform specific PI3K inhibitor (PI3Ki- α, PIK-75) overcomes resistance to Sorafenib. Indeed, while dual-targeting of PTENdeficient HCC cells with Sorafenib and PI3Ki-α results in synergistic growth inhibition, dual-targeting these cells with Sorafenib and a beta isoform specific inhibitor of PI3K (PI3Ki-α, TGX-221) leads to an antagonistic increase in tumor growth compared to single treatment with Sorafenib, since inhibiting p110-! promotes compensatory signaling from p110-α. We also investigated the cytotoxic effects of inhibiting Akt kinase by using an Akt2 isoform-specific inhibitor (Akti-2) and a general Akt inhibitor targeting both isoforms (Akti-1,2). Akti-1,2 and Akti-2 did not induce anti-growth or pro-apoptotic mechanisms, but they were highly effective in reducing migration. Akt2 isoform is specifically overexpressed in HCC, and is correlated with its progression. Moreover, the Akt2 isoform-specific role of Akt kinase on migration has been demonstrated in breast cancer, and depletion of AKT2, but not AKT1, was shown to promote regression of PTEN-deficient prostate cancer xenografts. Based on these findings, we predicted a prominent role of Akt2 in PTEN-deficient HCC cells and examined the therapeutic efficacy of combined treatment of Sorafenib and Akti-2 in vivo in athymic mice bearing Mahlavu tumor xenografts. After 3 weeks of treatment, tumor growth was reduced significantly in all tested groups (Sorafenib, Akti-2 and Combination) compared to the control group. Substantial intra-tumoral necrosis produced a temporary increase in tumor size but resulted in significant reduction in tumor weight (p<0.001) in combination-treated mice compared to Sorafenib-treated mice and further produced tumor regressions. In this study, we showed cytotoxic activity of a new PI3K α soform specific kinase inhibitor (PI3Ki-α / PIK-75) at 0.1 µM that acts synergistically with Sorafenib in vitro, determined the predominant role of PI3K isoform p110α in PTEN-deficient HCC cells, and revealed synergistic anti-tumor effect of combining Sorafenib with a new Akt isoform 2 specific kinase inhibitor (Akti-2) in vitro and in vivo.
Open Access
Targeted metabolomics revealed a key metabolic reprogramming in cholesterol biosynthesis pathway upon PTEN re-expression in PTEN-null, metastatic and castration-resistant prostate cancer
(2022-06) Güngül, Taha Buğra
Prostate cancer is the second most diagnosed type of cancer in males worldwide. Androgen signaling is a main driver of prostate cancers progression and androgen-deprivation therapies (ADT) are remained to be main treatment for preventing progression of the disease. Although ADT is effective at the first line and prolongs overall survival of the patients, eventually disease is recurred, develop resistance to castration and grow in an androgen-independent state, which completely eliminate ADT option. Metastatic and castration-resistant prostate cancers (mCPRC) are the most fatal type of the disease without any effective treatments currently, which is why, molecular drivers that contribute to emergence of castration-resistant phenotype need to be elucidated in order to develop efficient therapeutics against them. Metabolic reprogramming is one of the crucial hallmarks of cancer and loss-of tumor suppressor PTEN is an early and a frequent genetic alteration in prostate cancers, which leads to a hyperactivation of PI3K/Akt/mTOR axis and affects cellular metabolism widely. In this study, we aimed to unveil changes in the metabolome of C4-2 cells which are the type of mCRPC with a PTEN-null genetic background and target metabolic vulnerabilities of these cells. In order to address the question, we employed high-throughput metabolomics assay and revealed changes on metabolome of the cells upon re-expression of PTEN. We found that PTEN re-expression impaired the sphingolipid and cholesterol biosynthesis pathways of C4-2 cells. Upon PTEN re-expression, metabolism of C4-2 cells had tendency to increase the level of anti-survival metabolite; ceramide, and decrease pro-survival metabolite; sphingosine-1-phosphate. In addition to that, PTEN re-expression significantly impaired and downregulated the cholesterol metabolism of these cells. To target these metabolic vulnerabilities, we combined inhibitors of sphingolipid and cholesterol metabolisms with FDA-approved androgen antagonist, MDV3100, to determine possible synergistic effects from the combination of drugs. MDV3100 single treatment had only cytostatic effect on viability of C4-2 cells and combination of simvastatin, cholesterol metabolism inhibitor, with MDV3100 significantly decreased the cellular viability and resulted in significant synergistic effects in inhibiting the growth of C4-2 cells. Thus targeting cholesterol pathway in combination with androgen-deprivation therapies would be a promising approach to develop new combinatorial therapies and combat mCRPC.