Androgen receptor targeting drugs in castration-resistant prostate cancer and mechanisms of resistance.

Reactivated androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC). The novel AR targeting drugs abiraterone and enzalutamide have improved survival of CRPC patients. However, resistance to these agents develops and patients ultimately succumb to CRPC. Potential mechanisms of resistance include the following: 1) Expression of AR splice variants, such as the AR-V7 isoform, which lacks the ligand-binding domain; 2) AR missense mutations in the ligand-binding domain, such as F876L and T877A; and 3) Mutation or overexpression of androgen biosynthetic enzymes or glucocorticoid receptor. Several novel agents may overcome resistance mechanisms. Galeterone acts through multiple mechanisms that include degradation of AR protein and is being evaluated in CRPC patients positive for AR-V7. EPI-001 and related compounds inhibit AR splice variants by targeting the N-terminal transactivation domain of AR. Promising therapies and novel biomarkers, such as AR-V7, may lead to improved outcomes for CRPC patients.

Dasatinib inhibits site-specific tyrosine phosphorylation of androgen receptor by Ack1 and Src kinases.

Activation of androgen receptor (AR) may have a role in the development of castration-resistant prostate cancer. Two intracellular tyrosine kinases, Ack1 (activated cdc42-associated kinase) and Src, phosphorylate and enhance AR activity and promote prostate xenograft tumor growth in castrated animals. However, the upstream signals that activate these kinases and lead to AR activation are incompletely characterized. In this study, we investigated AR phosphorylation in response to non-androgen ligand stimulation using phospho-specific antibodies. Treatment of LNCaP and LAPC-4 cells with epidermal growth factor (EGF), heregulin, Gas6 (ligand binding to the Mer receptor tyrosine kinase and activating Ack1 downstream), interleukin (IL)-6 or bombesin stimulated cell proliferation in the absence of androgen. Treatment of LNCaP and LAPC-4 cells with EGF, heregulin or Gas6 induced AR phosphorylation at Tyr-267, whereas IL-6 or bombesin treatment did not. AR phosphorylation at Tyr-534 was induced by treatment with EGF, IL-6 or bombesin, but not by heregulin or Gas6. Small interfering RNA-mediated knockdown of Ack1 or Src showed that Ack1 mediates heregulin- and Gas6-induced AR Tyr-267 phosphorylation, whereas Src mediates Tyr-534 phosphorylation induced by EGF, IL-6 and bombesin. Dasatinib, a Src inhibitor, blocked EGF-induced Tyr-534 phosphorylation. In addition, we showed that dasatinib also inhibited Ack1 kinase. Dasatinib inhibited heregulin-induced Ack1 kinase activity and AR Tyr-267 phosphorylation. In addition, dasatinib inhibited heregulin-induced AR-dependent reporter activity. Dasatinib also inhibited heregulin-induced expression of endogenous AR target genes. Dasatinib inhibited Ack1-dependent colony formation and prostate xenograft tumor growth in castrated mice. Interestingly, Ack1 or Src knockdown or dasatinib did not inhibit EGF-induced AR Tyr-267 phosphorylation or EGF-stimulated AR activity, suggesting the existence of an additional tyrosine kinase that phosphorylates AR at Tyr-267. These data suggest that specific tyrosine kinases phosphorylate AR at distinct sites and that dasatinib may exert antitumor activity in prostate cancer through inhibition of Ack1.

The PTEN tumor suppressor is a negative modulator of androgen receptor transcriptional activity.

To investigate whether the tumor suppressor gene PTEN affects the activity of the androgen receptor (AR), we monitored the expression of the apoptotic gene HA-Bax (inserted in an adenovirus where it is driven by the AR-responsive promoter ARR(2)PB) in the presence or absence of dihydrotestosterone, in PTEN (+) or (-) prostate cancer cell lines, infected with an adenovirus containing wild-type PTEN (Av-CMV-PTEN) or a control LacZ-expressing construct. Our results showed that AR transcriptional activity was antagonized by PTEN expression. This antagonism was not cell line dependent, as it was observed in both LNCaP and LAPC-4 cells, or promoter dependent, as it was observed for a reporter gene (HA-Bax) driven by an exogenous androgen-responsive promoter (the ARR(2)PB promoter), and for a native gene (prostate-specific antigen; PSA) driven by an endogenous AR-responsive promoter. Additional experiments performed with viruses containing constitutively active (Adeno-myrAkt) or dominant negative (Adeno-dnAkt) forms of Akt demonstrated that Akt, a protein kinase whose activation is known to be inhibited by PTEN, mediated the observed antagonism between PTEN and AR transcriptional activity. Recently, two putative Akt phosphorylation sites have been identified in the AR sequence. Site-directed mutagenesis was utilized to convert these two serine into alanine residues. The resulting construct, named CMV-AR S213A&S791A was transfected in AR (-) and PTEN (-) PC-3 cells in the presence or absence of Av-CMV-PTEN and of two reporter plasmids (GRE(2)E1b-Luc and PSA P/E-luc) containing the luciferase gene driven by well-characterized androgen responsive promoters. These experiments demonstrated that, similarly to the wild-type molecule, AR S213A&S791A was transcriptionally inhibited by PTEN, suggesting that Akt does not have an effect on AR transcription by direct phosphorylation, but probably by affecting the availability of a downstream molecule whose main mechanism of action is that of modulating AR transcription. The data presented here suggest that loss of PTEN function may facilitate activation of AR signaling and progression to androgen independence in prostate cancer.

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency.

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G(1)/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2.

PTEN is a tumor suppressor gene mutated in human cancers. Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif. Using the yeast two-hybrid system, we isolated a membrane-associated guanylate kinase family protein with multiple PDZ domains [AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)]. MAGI-2 contains eight potential protein-protein interaction domains and is localized to tight junctions in the membrane of epithelial cells. PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2. MAGI-2 enhances the ability of PTEN to suppress Akt activation. Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein. We propose that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane.

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway.

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.

Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression.

The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription-PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.