Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage

Background: In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage. Methods: We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage. Results: A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420-520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420-520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency. Conclusions: USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420-520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.

Molecular mechanisms underpinning IFN-gamma and TNF-alpha synergy in intestinal epithelial cells

Cytokine-driven signalling shapes immune homeostasis and guides inflammatory responses mainly through induction of specific gene expression programmes both within and outside the immune cell compartment. These transcriptional outputs are often amplified via cytokine synergy, which sets a stimulatory threshold that safeguards from exacerbated inflammation and immunopathology. In this study, we investigated the molecular mechanisms underpinning synergy between two pivotal Th1 cytokines, IFN-γ and TNF-α, in human intestinal epithelial cells. These two proinflammatory mediators induce a unique state of signalling and transcriptional synergy implicated in processes such as antiviral and antitumour immunity, intestinal barrier and pancreatic β-cell dysfunction. Since its discovery more than 30 years ago, this biological phenomenon remains, however, only partially defined. Here, using a functional genomics approach including RNAi perturbation screens and small-molecule inhibitors, we identified two new regulators of IFN-γ/TNF-α-induced chemokine and antiviral gene and protein expression, a Bcl-2 protein BCL-G and a histone demethylase UTX. We also discovered that IFN-γ/TNF-α synergise to trigger a coordinated shutdown of major receptor tyrosine kinases expression in colon cancer cells. Together, these findings extend our current understanding of how IFN-γ/TNF-α synergy elicits qualitatively and quantitatively distinct outputs in the intestinal epithelium. Given the well-documented role of this synergistic state in immunopathology of various disorders, our results may help to inform the identification of high quality and biologically relevant druggable targets for diseases characterised by an IFN-γ/TNF-α high immune signature

Proteasome inhibitors in cancer therapy

The ubiquitin proteasome pathway plays a critical role in regulating many processes in the cell which are important for tumour cell growth and survival. Inhibition of proteasome function has emerged as a powerful strategy for anti-cancer therapy. Clinical validation of the proteasome as a therapeutic target was achieved with bortezomib and has prompted the development of a second generation of proteasome inhibitors with improved pharmacological properties. This review summarises the main mechanisms of action of proteasome inhibitors in cancer, the development of proteasome inhibitors as therapeutic agents and the properties and progress of next generation proteasome inhibitors in the clinic.

Pathogenesis of prostate cancer and hormone refractory prostate cancer

Prostate cancer is the second most common malignancy in males and the leading cause of cancer death. Prostate cancer is initially androgen dependent and relies upon the androgen receptor (AR) to mediate the effects of androgens. The AR is also the target for therapy using antiandrogens and LHRH analogues. However, all cancers eventually become androgen independent, often referred to as hormone refractory prostate cancer. The processes involved in this transformation are yet to be fully understood but research in this area has discovered numerous potential mechanisms including AR amplification, over-expression or mutation and alterations in the AR signaling pathway. This review of the recent literature examines the current knowledge and developments in the understanding of the molecular biology of prostate cancer and hormone refractory prostate cancer, summarizing the well characterized pathways involved as well as introducing new concepts that may offer future solutions to this difficult problem.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Adhésion thérapeutique aux traitements oncologiques oraux et prise en charge interdisciplinaire [Therapeutic adherence to oral cancer therapy and interdisciplinary management].

Medication adherence is a well-known risk factor in internal medicine. However in oncology this dimension is emerging due to the increasing number of oral formulations. First results in the oral oncology literature suggest that patients' ability to cope with medical prescription decreases with time. This might preclude patients from reaching clinical outcomes. Factors impacting on medication adherence to oral oncology treatments have not been yet extensively described neither strategies to address them and support patient's needs. Oncologists and pharmacists in our University outpatient settings performed a pilot study which aimed at measuring and facilitating adherence to oral oncology treatments and at understanding determinants of patient's adherence. The ultimate purpose of such a patient-centered and interdisciplinary collaboration would be to promote patient self-management and complement the standard medical follow-up.

Molecular mechanisms controlling the precision of chemokine guided cell migration

By the end of the first day of embryonic development, zebrafish primordial germ cells (PGCs) arrive at the site where the gonad develops. In our study we investigated the mechanisms controlling the precision of primordial germ cell arrival at their target. We found that in contrast with our expectations which were based on findings in Drosophila and mouse, the endoderm does not constitute a preferred migration substrate for the PGCs. Rather, endoderm derivatives are important for later stages of organogenesis keeping the PGC clusters separated. It would be interesting to investigate the precise mechanism by which endoderm controls germ cell position in the gonad. In their migration towards the gonad, zebrafish germ cells follow the gradient of chemokine SDF-1a, which they detect using the receptor CXCR4b that is expressed on their membrane. Here we show that the C-terminal region of CXCR4b is responsible for down-regulation of receptor activity as well as for receptor internalization. We demonstrate that receptor molecules unable to internalize are less potent in guiding germ cells to the site where the gonad develops, thereby implicating chemokine receptor internalization in facilitating precision of migration during chemotaxis in vivo. We demonstrate that while CXCR4b activity positively regulates the duration of the active migration phases, the down-regulation of CXCR4b signalling by internalization limits the duration of this phase. This way, receptor signalling contributes to the persistence of germ cell migration, whereas receptor down-regulation enables the cells to stop and correct their migration path close to the target where germ cells encounter the highest chemokine signal. Chemokine receptors are involved in directing cell migration in different processes such as lymphocyte trafficking, cancer and in the development of the vascular system. The C-terminal domain of many chemokine receptors was shown to be essential for controlling receptor signalling and internalization. It would therefore be important to determine whether the role for receptor internalization in vivo as described here (allowing periodical corrections to the migration route) and the mechanisms involved (reducing the level of signalling) apply for those other events, too.

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Flavonoids as prospective compounds for anti-cancer therapy

Flavonoids, which are polyphenolic compounds, are a class of plant secondary metabolites possessing a broad spectrum of pharmacological activity including anti-cancer activities. They have been reported to interfere in the initiation, promotion and progression of cancer by modulating different enzymes and receptors in signal transduction pathways related to cellular proliferation, differentiation, apoptosis, inflammation, angiogenesis, metastasis and reversal of multidrug resistance. Due to their multiple molecular mechanisms of action, flavonoids (both natural and synthetic analogs) are being investigated for their potential applications in anti-cancer therapies. In this review article, the main molecular mechanisms of action of flavonoids attributing to their potential anti-cancer activities have been discussed and the key structural features required for their activity are highlighted.

Use of grape polyphenols against carcinogenesis: Putative molecular mechanisms of action using in vitro and in vivo test systems

Polyphenols are present in foods and beverages and are related to sensorial qualities such as color, bitterness, and astringency, which are relevant in wine, tea, grape juice, and other products. These compounds occur naturally in forms varying from simple phenolic acids to complex polymerized tannins. Thus, it is reasonable to expect that grape-derived products elaborated in the presence of skins and seeds, such as wine and grape juice, are natural sources of flavonoids in the diet. Carcinogenesis is a multistep process that is characterized by genetic, epigenetic, and phenotypic changes. With increasing knowledge of these mechanisms, and the conclusion that most cases of cancer are preventable, efforts have focused on identifying the agents with potential anticancer properties. The use of grape polyphenols against the carcinogenesis process seems to be a suitable alternative for either prevention and/or therapeutic purposes. The aim of this article is to show the molecular data generated from the use of grape polyphenols against carcinogenesis using in vivo and in vitro test systems. © Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition.

Cancer therapy-associated cardiotoxicity and signaling in the myocardium

The cardiotoxic potential of cytotoxic cancer chemotherapy is well known. Prime examples are the anthracyclines, which are highly efficacious agents for hemopoietic malignancies and solid tumors, but their clinical use is limited primarily by cardiotoxicity. Besides the conventional chemotherapeutics, new cancer drugs were developed in the last decade with the goal to specifically inhibit selected molecular targets such as growth factor receptors or intracellular tyrosine kinases in cancer cells. However, the outcome of combining conventional and newer cancer therapies could have unexpected side effects not anticipated so far and the long-term outcome is not known. Sometimes, however, unexpected side effects also shed light on previously unknown physiological functions. For example, the anti-HER2 cancer therapeutic trastuzumab (Herceptin), which can induce cardiac dysfunction, has demonstrated the importance of the ErbB/neuregulin signaling system in the adult heart. Subsequently, the role of endothelial-myocardial communication in maintaining phenotype and survival of adult cardiomyocytes has increasingly been recognized.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

MOLECULAR MECHANISMS UNDERLYING THE TRANSCRIPTIONAL REGULATION OF T HELPER 17 AND REGULATORY T CELLS

CD4+ T helper (Th) lymphocytes are vital for integrating immune responses by orchestrating the function of other immune cell types. Naïve Th cells can differentiate into different effector subsets that are characterized by their cytokine profile and immune regulatory functions. These subsets include Th1, Th2, Th17, natural and inducible regulatory T cells (nTreg and iTreg respectively), among others. We focused our investigation on two Th lineages, Th17 and regulatory T cells, with opposing functions in the immune system. These subsets have been suggested to be reciprocally regulated since they both require TGF-b for their development. We investigated the role of the Treg-associated master transcription factor Foxp3, and found that Foxp3 inhibits Th17 cell generation by preventing the transcriptional activity of the two main Th17-specific transcription factors, nuclear orphan receptors RORa and RORgt. At the molecular level, we identified two different functional domains in Foxp3 required for such inhibition: the LQALL sequence in exon 2 and the TIP60/HDAC7 binding domain. These domains could be crucial to either prevent the association of the nuclear receptors to coactivators or to recruit histone deacetylases to RORa- or RORgt-target genes. Since TGF-b is a common cytokine required for the commitment towards both Th lineages, we determined the role of the TGF-b-dependent signaling pathway in the generation of each subset. By using mice with deficiencies in signaling molecules downstream of TGF-b, we found that while Smad2, Smad3 and Smad4 are required for the generation of iTreg cells, only Smad2 is indispensable for the induction of IL-17-producing cells, suggesting that TGF-b induces these T helper lineages through differential signaling pathways. Thus, our findings describe novel transcriptional regulatory mechanisms that control the generation of two T helper lineages with opposing functions. These findings could provide novel therapeutic targets to treat diseases where the balance of these T cells is dysregulated, such as in autoimmunity, chronic infectious diseases and cancer.