High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma

The phosphatidylinositol 3'-kinase (PI3K) pathway is activated in many human cancers. In addition to inactivation of the PTEN tumor suppressor gene, mutations or amplifications of the catalytic subunit alpha of PI3K (PIK3CA) have been reported. However, the coexistence of mutations in these two genes seems exceedingly rare. As PTEN mutations occur at high frequency in endometrial carcinoma, we screened 66 primary endometrial carcinomas for mutations in the helical and catalytic domains of PIK3CA. We identified a total of 24 (36%) mutations in this gene and coexistence of PIK3CA/PTEN mutations at high frequency (26%). PIK3CA mutations were more common in tumors with PTEN mutations (17 of 37, 46%) compared with those without PTEN mutations (7 of 29, 24%). Array comparative genomic hybridization detected 3q24-qter amplification, which covers the PIK3CA gene (3q26.3), in one of nine tumors. Knocking down PTEN expression in the HEC-1B cell line, which possesses both K-Ras and PIK3CA mutations, further enhances phosphorylation of Akt (Ser473), indicating that double mutation of PIK3CA and PTEN has an additive effect on PI3K activation. Our data suggest that the PI3K pathway is extensively activated in endometrial carcinomas, and that combination of PIK3CA/PTEN alterations might play an important role in development of these tumors.     

PTEN抑癌基因异常与肿瘤靶向治疗

PTEN在肿瘤中存在基因组、基因转录、翻译和翻译后等多个水平的异常,从而导致PI3K/Akt通路的过度激活.这不仅是肿瘤发生的重要机制,也与某些靶向治疗后的耐药相关.克服PTEN异常所致耐药的方法包括直接应用作用于P13K/Akt通路的新型靶向药物以及重新恢复PTEN基因的表达和功能.     

Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

Introduction

Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.

Methods

In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines.

Results

The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition.

Conclusions

These data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.     

Preferential killing of PTEN-null myelomas by PI3K inhibitors through Akt pathway

We recently reported that internal deletion of PTEN tumor suppressor gene in OPM2 and Delta47 myeloma lines led to high Akt activation. Re-expression of PTEN induced strong apoptosis and growth inhibition. To understand the biologic importance of the phosphatidylinositol 3 kinase (PI3K)/Akt activation affected by PTEN deletion, we analysed apoptosis and growth inhibition by applying PI3K inhibitors to myeloma lines and by expressing Akt constructs. The PI3K inhibitors preferentially suppressed PTEN-null myeloma growth to those expressing PTEN, indicating that PI3K activation is more critical for growth and survival of those lines with PTEN mutations than others expressing a functional PTEN gene. Since PTEN-null myeloma lines exhibited much stronger Akt activation than PTEN-expressing cells in response to insulin-like growth factor I stimulation, we determined whether Akt could be responsible for PI3K-mediated cell survival and growth of PTEN-null myeloma lines. Expression of an active Akt, but not its kinase dead mutant, reversed wortmannin- and dexamethasone-induced apoptosis and growth inhibition in PTEN-null myeloma lines, suggesting that Akt lies downstream of PI3K for PTEN-null myeloma survival and dexamethasone resistance. In summary, we have provided evidence that PTEN-null myeloma cells are stringently dependent on the PI3K/Akt activation for cell survival. These results may provide a basis to treat myeloma patients with PI3K and Akt inhibitors.     

Mechanisms of regulation of cell adhesion and motility by insulin receptor substrate-1 in prostate cancer cells

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate-1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these effects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin alpha 5 beta 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating effects that are different, and even opposite, from those generated by IGF-I.     

PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death

PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.     

PTEN’s regulation of VEGF and VEGFR1 expression and its clinical significance in myeloid leukemia

Phosphatase and tensin homolog (PTEN) acts as a novel tumor suppressor gene. PTEN protein plays an important role in regulating proliferation, apoptosis, invasion, and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating PI3K/Akt pathway in many solid tumors. However, the effects of PTEN on VEGF and VEGFR1 (FLT1)-mediated angiogenesis, migration, invasion of leukemia cells, and its clinical significance are still unknown in myeloid leukemia. Therefore, we investigated the effect of PTEN on PI3K/Akt and VEGF/FLT1 pathways by transfecting wild-type PTEN gene to induce high expression of wild-type PTEN gene and protein in chronic myelogenous leukemia cell line K562 cells. We also observed the correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients. We found that the expression reconstitution of wild-type PTEN had significant effect on inhibiting proliferation, migration, and invasion abilities of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Down-regulation of PTEN expression accompanied by up-regulation of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. In conclusion, PTEN could modulate the function of VEGF/VEGFR signaling pathway down-regulation of Akt phosphorylation and that PTEN would be a candidate target to be addressed for inhibiting angiogenesis along with the treatment of myeloid leukemia.     

Drug discovery approaches targeting the PI3K/Akt pathway in cancer

The abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been validated by epidemiological and experimental studies as an essential step toward the initiation and maintenance of human tumors. Notable in this regard are the prevalent somatic genetic alterations leading to the inactivation of the tumor suppressor gene PTEN and gain-of-function mutations targeting PIK3CA--the gene encoding the catalytic phosphosinositide-3 kinase subunit p110 alpha. A number of the intracellular components of this pathway have been targeted as anticancer drug discovery activities leading to the current panoply of clinical trials of inhibitors of PI3K, Akt and HSP90 in man. This review summarizes current preclinical knowledge of modulators of the PI3K/Akt pathway in which drug discovery and development activities have been advanced focusing on both the relevant clinical stage inhibitors and other disclosed tool compounds targeting PI3K, PDK1, Akt and HSP90.     

PTEN,but not SHIP and SHIP2, suppresses the PI3K/Akt pathway and induces growth inhibition and apoptosis of myeloma cells

Expression of PTEN tumor suppressor gene has been known to dephosphorylate the phosphatidylinositol 3' kinase (PI3K) products on the 3 prime inositol ring, resulting in reduced Akt activation. Loss of PTEN expression in OPM2 and delta47 human myeloma lines led to high Akt activity toward insulin-like growth factor I (IGF-I). In contrast, mouse plasma cell tumor (PCT) lines, expressing wild type PTEN, did not respond to IGF-I for Akt activation. We demonstrated here that endogenous PTEN played a negative role in controlling Akt activity in both mouse PCT and NIH3T3 fibroblast lines by using anti-sense oligonucleotides against PTEN. To determine the role of src-homology 2-containing inositol 5' phosphatase (SHIP) in regulating the PI3K/Akt pathway, we manipulated its expression by down-regulation and overexpression in myeloma, PCT and NIH3T3 lines and analysed Akt activation. Our results showed that SHIP, unlike PTEN, did not affect Akt activity in all systems analysed, despite its ability to dephosphorylate a PI3K product. Although SHIP2 expression resulted in suppression of interleukin-6-mediated mitogen-activated protein kinase activation, expression of SHIP and SHIP2 in a PTEN-null myeloma line did not suppress Akt activity. Biologically, expression of only PTEN, but not SHIP and SHIP2, resulted in growth inhibition and increased apoptosis in OPM2 myeloma line. Together, our results have established the role of PTEN, but not SHIP and SHIP2, in negatively regulating the PI3K/Akt cascade and in myeloma leukemogenesis.     

Inhibition of phosphatidylinositol 3-kinase signaling negates the growth advantage imparted by a mutant epidermal growth factor receptor on human glioblastoma cells

In de novo glioblastoma multiforme, loss of the tumour suppressor protein PTEN can coincide with the expression of a naturally occurring mutant epidermal growth factor receptor known as deltaEGFR. DeltaEGFR signals constitutively via the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt and mitogen-activated protein kinase pathways. In human U87MG glioblastoma cells that lack PTEN, deltaEGFR expression enhances tumourigenicity by increasing cellular proliferation. Inhibition of PI3K signaling with the pharmacologic inhibitor wortmannin, or by the reconstitution of physiological levels of PTEN to dephosphorylate the lipid products of PI3K, negated the growth advantage imparted by deltaEGFR on U87MG cells. PTEN reconstitution suppressed the elevated PI3K signaling, without affecting mitogen-activated protein kinase signaling and caused a delay in G1 cell cycle progression that was concomitant with increased cyclin-dependent protein kinase inhibitor p21CIP1/WAF1 protein levels. Our study provides insight into the mechanism by which deltaEGFR may contribute to glioblastoma development.     

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.     

Akt regulates COX-2 mRNA and protein expression in mutated-PTEN human endometrial cancer cells

In human endometrial cancer, the fourth most common cancer in women, tumor suppressor phosphatase tensin homologue (PTEN) is frequently mutated. In the presence of a mutated PTEN protein, Akt phosphorylation levels are increased leading to the activation of this survival pathway. Numerous studies indicated that COX-2 is inappropriately induced and up-regulated in a number of malignant cancer cells. COX-2 plays an important role in tumor cell biology, taking part actively in angiogenesis particularly via the production of prostaglandin E2 (PGE2). The present study was undertaken to determine the involvement of PI 3-K/Akt pathway in the regulation of COXs expression and PGE2 synthesis. Three different human endometrial cancer cell lines known to have wild-type PTEN (HEC 1-A) or a mutated inactive PTEN protein (RL 95-2 and Ishikawa) were used for these studies. Results showed that Akt phosphorylation was high in mutated PTEN cells. RT-PCR studies revealed that Akt1 and Akt2 were the regulated forms whereas Akt3 mRNA was nearly undetectable. COX-2 mRNA expression and protein levels were high in these cells compared to wild-type PTEN cells as demonstrated by RT-PCR and Western analysis respectively. PGE2 production was higher in mutated-PTEN expressing phospho-Akt and COX-2 compared to wild-type PTEN cells. Inhibition of PI 3-K with Wortmannin and LY294002 blocked Akt phosphorylation and inhibited expression of COX-2 in mutated-PTEN cells. Inhibition of Akt phosphorylation with specific PI 3-K inhibitors and down-regulation of COX-2 increased apoptosis in human endometrial cancer cells. Likewise, transfection of mutated-PTEN cells with a dominant negative Akt vector, resulted in COX-2 down-regulation and activation of apoptosis, as demonstrated by Hoechst nuclear staining. On the opposite, activation of Akt using a constitutively active expression vector, resulted in the up-regulation of COX-2 protein expression. Specific inhibition of COX-2 with NS-398 induced apoptosis in COX-2 expressing human endometrial cancer cells. It is concluded that the PI 3-K/Akt survival pathway is involved in the regulation of COX-2 and PGE2 synthesis in human endometrial cancer cells.     

Inhibition of ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and delays tumor growth

The tumor suppressor gene phosphatase and tensin homologue (PTEN) regulates the phosphatidylinositol-3'-kinase (PI3K) signaling pathway and has been shown to correlate with poor prognosis in high-grade astrocytomas when mutational inactivation or loss of the PTEN gene occurs. PTEN mutation leads to constitutive activation of protein kinase B (PKB)/Akt with phosphorylation at the PKB/Akt sites Thr-308 and Ser-473. Integrin-linked kinase (ILK) has been shown to regulate PKB/Akt activity with the loss of PTEN in prostate cancer. We now demonstrate that ILK activity regulates PKB/Akt activity in glioblastoma cells. The activity of ILK is constitutively elevated in a serum-independent manner in PTEN mutant cells, and transfection of wild-type PTEN under the control of an inducible promoter into mutant PTEN cells inhibits ILK activity. Transfection of ILK antisense (ILKAS) or exposure to a small-molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473 in PTEN-mutant glioblastoma cell lines. In addition, the delivery of ILKAS to PTEN-negative glioblastoma cells resulted in apoptosis. Rag-2M mice bearing established ( approximately 100 mg) human U87MG glioblastoma tumors, treated QD x 5 for 3 consecutive weeks with ILKAS (i.p. 5 mg/kg), exhibited stable disease with < or =7% increase in tumor volume over the 3-week course of treatment. In contrast, animals treated with an oligonucleotide control or saline exhibited a >100% increase in tumor volume over the same time period. Our initial results indicate that therapeutic strategies targeting ILK may be beneficial in the treatment of glioblastomas.     

Akt activity in endometrial cancer cells: regulation of cell survival through cIAP-1

In a number of different cancer including endometrial cancers, tumor suppressor phosphatase tensin homologue (PTEN, a lipid phosphatase) is frequently mutated. PTEN dephosphorylates PI 3-K product, phosphatidylinositol 3,4,5-triphosphate (PIP3), into inactive PIP2 which blocks Akt activation/phosphorylation. In the present study, we have used an endometrial cancer cell line known to possess wild-type PTEN (HEC-1-A) and two mutated inactive PTEN protein cell lines (RL-95-2 and Ishikawa) to investigate importance of PI 3-K/PTEN/Akt survival pathway in endometrial cancers. As hypothesised, results showed high levels of Akt1/2 mRNAs and protein phosphorylation in the two mutated PTEN human endometrial cancer cells. To test the possible involvement of Akt in the regulation of survival factors, Bcl-2, XIAP, cIAP-1 and cIAP-2 expression were measured. cIAP-1 protein expression was high in cells expressing phospho-Akt. XIAP and cIAP-2 protein expression was not influenced by the presence of active Akt. Akt phosphorylation decreased and apoptosis was strongly increased in mutated PTEN human endometrial cancer cells in the presence of PI 3-K inhibitor (Wortmannin) which was accompanied by a down-regulation of cIAP-1 protein. Wortmannin had no effect on wild-type PTEN HEC-1-A cell line. Although, Bcl-2 expression was strongly expressed in mutated-PTEN cells, expression remained stable in the presence of Wortmannin suggesting that Bcl-2 is not regulated by Akt. Overexpression of Akt using a constitutively active Akt expression vector resulted in an up-regulation of cIAP-1 expression. These results suggest a pivotal role of Akt in the regulation of endometrial cancer cell survival through the up-regulation of a specific inhibitor of apoptosis protein.     

Correlation between loss of PTEN expression and Akt phosphorylation in endometrial carcinoma.

The tumor suppressor PTEN acts as a lipid phosphatase, regulates the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway, and modulates cell cycle progression and cell survival. Somatic mutations of PTEN have been reported in a variety of cancers, especially in endometrial carcinoma. To clarify whether and how PTEN and the PI3K/Akt pathway relates to endometrial carcinoma, we examined the expression of those pathway-related proteins in patients with endometrial carcinoma. Of 103 endometrial carcinomas, 37 (36%) showed negative immunohistochemical staining of PTEN. Western blotting revealed that the expression of PTEN in PTEN-negative cases was significantly lower compared with that in positive cases. In contrast, phospho-Akt level in negative cases was significantly higher. We found a significant inverse correlation between PTEN and phospho-Akt (r = -0.796). The expression of phospho-Bad was greater in negative cases, suggesting that Bad might be a target for AKT: The present study demonstrates the phosphorylation of Akt accompanied by the loss of PTEN in clinical specimens of endometrial carcinomas.     

Levels of PTEN protein modulate Akt phosphorylation on serine 473, but not on threonine 308, in IGF-II-overexpressing rhabdomyosarcomas cells

Constitutive activation of Akt has been found in many types of human cancer, and is believed to promote proliferation and increased cell survival thereby contributing to cancer progression. In this study, we examined Akt phosphorylation on Ser473 and Thr308 in seven IGF-II-overexpressing rhabdomyosarcomas (RMS) cells. All the RMS cell lines tested had high levels of Akt phosphorylation on Thr308, whereas three cell lines (Rh5, Rh18, and CTR) had a much lower level of Akt phosphorylation on Ser473. To determine whether the difference in Akt phosphorylation on Ser473, but not on Thr308, observed among cell lines is a cell-specific phenomenon or due to other factors, which possibly downregulate Akt phosphorylation, we examined expression of PTEN protein, which acts as a negative regulator of the PI3K/Akt signaling pathway through its ability to dephosphorylate phosphatidylinositol 3,4,5-triphosphate (PIP3). The levels of PTEN expression inversely correlate with Akt phosphorylation on Ser473, but not on Thr308. Consistent with this finding, transfection of wild-type PTEN into RMS and mouse myoblast C2C12 cells resulted in reduced Akt phosphorylation on Ser473, but not on Thr308. Our data suggest that Ser473 may be a key target residue for PTEN to modulate the effects of IGF-II on activating the PI3K/Akt pathway in RMS cells. A better understanding of the pathway in RMS will likely contribute to insights into the biology of the RMS tumorigenesis and hopefully lead to novel therapeutic options.     

Dimethylsulfoxide induces upregulation of tumor suppressor protein PTEN through nuclear factor-kappaB activation in HL-60 cells

Dimethylsulfoxide (DMSO) has been known to differentiate HL60 cells into neutrophil like cells. Here, we provide an evidence for the involvement of tumor suppressor PTEN, an antagonist of phosphatidylinositol 3-kinase (PI3K) in the DMSO-induced differentiation of HL60 cells. DMSO upregulated PTEN with unaffecting the expression of PI3K. The upregulation of PTEN by DMSO lead to the decrease of Akt phosphorylation, a downstream of PI3K. The DMSO-induced upregulation of PTEN might be mediated by NF-kappaB activation, which was evidenced by the blockage of DMSO-induced PTEN upregulation with an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC).     

Caffeine activates tumor suppressor PTEN in sarcoma cells

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Akt activation exerts a strong anti-apoptotic effect and inhibits key pro-apoptotic proteins. We investigated the effect of caffeine in the prevention of tumor cell proliferation and induction of cell death. We found that caffeine induced increased intracellular cAMP levels, PTEN activation and Akt inactivation, which together prevented proliferation of human osteosarcoma cells (MG63) and fibrosarcoma cells (HT1080). PTEN knockdown by siRNA reduced the effects of caffeine on Akt inactivation in osteosarcoma cells. These results indicate that the tumor suppressor PTEN signaling pathway contributes to the growth-inhibitory effect of caffeine on sarcoma cells. Our data suggest that caffeine and other drugs that act on this pathway could have promising therapeutic effects in the treatment of sarcoma patients.     

Enhanced cell growth inhibition following PTEN nonviral gene transfer using polyethylenimine and photochemical internalization in endometrial cancer cells

PTEN is a tumor suppressor gene mapped on chromosome 10q23.3 and encodes a dual specificity phosphatase. PTEN has major implication in PI3 kinase (PI3K) signal transduction pathway and negatively controls PI3 phosphorylation. It has been reported to be implicated in cell cycle progression and cell death control through inhibition of PI3K-Akt signal transduction pathway and in the control of cell migration and spreading through its interaction with focal adhesion kinase. Somatic mutations of PTEN are frequently detected in several cancer types including brain, prostate and endometrium with more than 30% of tumor tissue specimens bearing PTEN mutations and/or deletions. Because of its high frequency of mutations and its important function as tumor suppressor gene, PTEN is a good candidate for gene therapy. Inducible expression of PTEN has been also reported. In cancer cells bearing PTEN abnormalities, the reversion of PTEN function by external gene transfer becomes more and more investigated in cancer treatment research. Several technologies including the photochemical internalization (PCI) and aiming at improving the transfection efficiency have been reported. PCI is an innovative procedure based on light-induced delivery of macromolecules such as DNA, proteins and other therapeutic molecules from endocytic vesicles to the cytosol of target cells. PCI has been reported to enhance the gene delivery potential of viral and nonviral vectors. The present study was designed to evaluate the influence of photochemical internalization on polyethylenimine (PEI)-mediated PTEN gene transfer and its effects on the cellular viability in Ishikawa endometrial cancer cells bearing PTEN abnormalities. PCI was found to significantly (P < 0.01) enhance PTEN mRNA expression (4.2 fold increase). Subsequently, following PEI-mediated PTEN gene transfer, the restoration of the PTEN protein expression was observed. As a consequence, significant cell growth inhibition (44%) was observed in Ishikawa endometrial cells. Using PCI for PEI-mediated PTEN gene transfer was found to further enhance PTEN mRNA and protein expression as well as PTEN-related cell growth inhibition reaching 89%.