The kallikrein 14 gene is down-regulated by androgen receptor signalling and harbours genetic variation that is associated with prostate tumour aggressiveness

Kallikrein 14 (KLK14) has been proposed as a useful prognostic marker in prostate cancer, with expression reported to be associated with tumour characteristics such as higher stage and Gleason score. KLK14 tumour expression has also shown the potential to predict prostate cancer patients at risk of disease recurrence after radical prostatectomy. The KLKs are a remarkably hormone-responsive family of genes, although detailed studies of androgen regulation of KLK14 in prostate cancer have not been undertaken to date. Using in vitro studies, we have demonstrated that unlike many other prostatic KLK genes that are strictly androgen responsive, KLK14 is more broadly expressed and inversely androgen regulated in prostate cancer cells. Given these results and evidence that KLK14 may play a role in prostate cancer prognosis, we also investigated whether common genetic variants in the KLK14 locus are associated with risk and/or aggressiveness of prostate cancer in approximately 1200 prostate cancer cases and 1300 male controls. Of 41 single nucleotide polymorphisms assessed, three were associated with higher Gleason score (≥7): rs17728459 and rs4802765, both located upstream of KLK14, and rs35287116, which encodes a p.Gln33Arg substitution in the KLK14 signal peptide region. Our findings provide further support for KLK14 as a marker of prognosis in prostate cancer.     

Prediction and Prioritization of Rare Oncogenic Mutations in the Cancer Kinome Using Novel Features and Multiple Classifiers

Cancer is a genetic disease that develops through a series of somatic mutations, a subset of which drive cancer progression. Although cancer genome sequencing studies are beginning to reveal the mutational patterns of genes in various cancers, identifying the small subset of “causative” mutations from the large subset of “non-causative” mutations, which accumulate as a consequence of the disease, is a challenge. In this article, we present an effective machine learning approach for identifying cancer-associated mutations in human protein kinases, a class of signaling proteins known to be frequently mutated in human cancers. We evaluate the performance of 11 well known supervised learners and show that a multiple-classifier approach, which combines the performances of individual learners, significantly improves the classification of known cancer-associated mutations. We introduce several novel features related specifically to structural and functional characteristics of protein kinases and find that the level of conservation of the mutated residue at specific evolutionary depths is an important predictor of oncogenic effect. We consolidate the novel features and the multiple-classifier approach to prioritize and experimentally test a set of rare unconfirmed mutations in the epidermal growth factor receptor tyrosine kinase (EGFR). Our studies identify T725M and L861R as rare cancer-associated mutations inasmuch as these mutations increase EGFR activity in the absence of the activating EGF ligand in cell-based assays.     

Hereditary cancer-associated missense mutations in hMSH6 uncouple ATP hydrolysis from DNA mismatch binding

Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.     

Genetic Variants in CASP3, BMP5, and IRS2 Genes May Influence Survival in Prostate Cancer Patients Receiving Androgen-Deprivation Therapy

Several genome-wide association studies (GWAS) have been conducted to identify the common single nucleotide polymorphisms (SNPs) that influence the risk of prostate cancer. It was hypothesized that some prostate cancer-associated SNPs might relate to the clinical outcomes in patients treated for prostate cancer using androgen-deprivation therapy (ADT). A cohort of 601 patients who have received ADT for prostate cancer was genotyped for 29 SNPs that have been associated with prostate cancer in Cancer Genetic Markers of Susceptibility GWAS, and within the genes that have been implicated in cancer. Prognostic significance of these SNPs on the disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT were assessed by Kaplan-Meier analysis and Cox regression model. Three SNPs, namely CASP3 rs4862396, BMP5 rs3734444 and IRS2 rs7986346, were found to be closely associated with the ACM (P≤0.042), and BMP5 rs3734444 and IRS2 rs7986346 were also noted to be significantly related to the PCSM (P≤0.032) after adjusting for the known clinicopathologic predictors. Moreover, patients carrying a greater number of unfavorable genotypes at the loci of interest had a shorter time to ACM and PCSM during ADT (P for trend <0.001). Our results suggest that CASP3 rs4862396, BMP5 rs3734444 and IRS2 rs7986346 may affect the survival in patients after ADT for prostate cancer, and the analysis of these SNPs can help identify patients at higher risk of poor outcome.     

The significance of galectin-3 as a new basal cell marker in prostate cancer

Prostate cancer may originate from distinct cell types, resulting in the heterogeneity of this disease. Galectin-3 (Gal-3) and androgen receptor (AR) have been reported to play important roles in the progression of prostate cancer, and their heterogeneous expressions might be associated with different cancer subtypes. Our study found that in various prostate cancer cell lines Gal-3 expression was always opposite to AR expression and other luminal cell markers but consistent with basal cell markers including glutathione S-transferase-π and Bcl-2. This expression pattern was confirmed in human prostate cancer tissues. Our results also showed that prostate cancer cells positive with basal cell markers were more aggressive. Downregulation of Gal-3 expression resulted in increased apoptotic potential and decreased metastasis potential of prostate cancer cells. Our findings demonstrate for the first time that Gal-3 may serve as a new marker for basal characteristics of prostate cancer epithelium. This study helps us to better understand the heterogeneity of prostate cancer. The clinical significance of this study lies in the application of Gal-3 to distinguish prostate cancer subtypes and improve treatment efficacy with designed personalized therapy.     

Androgen deficiency in the aging male: benefits and risks of androgen supplementation

There is now convincing evidence that in a subset of aging men, increasing with age, plasma testosterone levels fall below a critical level resulting in hypogonadism. This state of testosterone deficiency has an impact on bone, muscle and brain function and is maybe a factor in the accumulation of visceral fat which again has a significant impact on the cardiovascular risk profile. From the above it follows that androgen replacement to selected men with proven androgen deficiency will have beneficial effects. There is, however a concern that androgen administration to aging men may be harmful in view of effects on prostate disease. Benign prostate hyperplasia (BPH) and prostate cancer are typically diseases of the aging male, steeply increasing with age. But epidemiological studies provide no clues that the levels of circulating androgen are correlated with or predict prostate disease. Similarly, androgen replacement studies in men do not suggest that these men suffer in a higher degree from prostate disease than control subjects. It seems a defensible practice to treat aging men with androgens if and when they are testosterone-deficient, but long-term studies including sufficient numbers of men are needed.     

Stromal Hyaluronan Interaction with Epithelial CD44 Variants Promotes Prostate Cancer Invasiveness by Augmenting Expression and Function of Hepatocyte Growth Factor and Androgen Receptor

The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of prostate cancer. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support tumor epithelial cell survival. Overexpression of hyaluronan, CD44v9 variants, and stroma-derived growth factors/cytokines are specific features in many cancers, including prostate cancer. Androgen/androgen receptor interaction has a critical role in regulating prostate cancer growth. Our previous study showed that 1) that increased synthesis of hyaluronan in normal epithelial cells promotes expression of CD44 variants; 2) hyaluronan interaction with CD44v6-v9 promotes activation of receptor-tyrosine kinase, which stimulates phosphatidylinositol 3-kinase-induced cell survival pathways; and 3) CD44v6/short hairpin RNA reduces colon tumor growth in vivo (Misra, S., Hascall, V. C., De Giovanni, C., Markwald, R. R., and Ghatak, S. (2009) J. Biol. Chem. 284, 12432–12446). Our results now show that hepatocyte growth factor synthesized by myofibroblasts associated with prostate cancer cells induces activation of HGF-receptor/cMet and stimulates hyaluronan/CD44v9 signaling. This, in turn, stabilizes the androgen receptor functions in prostate cancer cells. The stroma-derived HGF induces a lipid raft-associated signaling complex that contains CD44v9, cMet/phosphatidylinositol 3-kinase, HSP90 and androgen receptor. CD44v9/short hairpin RNA reverses the assembly of these components in the complex and inhibits androgen receptor function. Our results provide new insight into the hyaluronan/CD44v9-regulated androgen receptor function and the consequent malignant activities in prostate cancer cells. The present study describes a physiologically relevant in vitro model for studying the molecular mechanisms by which stroma-derived HGF and hyaluronan influence androgen receptor and CD44 functions in the secretory epithelia during prostate carcinogenesis.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

Prostate cancer: Re-focusing on androgen receptor signaling

Prostate cancer is the most common, non-dermatologic cancer in men. Since prostate cancer is highly associated with increased age, the incidence of this disease is expected to increase as the population ages. In its initial stages prostate cancer depends upon the actions of androgen, and androgen deprivation therapy induces tumor regression. Currently, androgen deprivation is achieved by either surgical or chemical androgen blockade. Unfortunately, nearly all prostate cancer patients develop tumors that grow despite androgen blockade and ultimately relapse. Many alterations in prostate cancer cells contribute to this state. Although chemotherapy induces short remissions in some patients, there are no curative therapies for metastatic disease. This review summarizes our current understanding in androgen signaling and the mechanisms that allow tumor cells to bypass androgen manipulation therapy. The identification of novel survival pathways and effector molecules that drive androgen independent growth is necessary to develop effective therapies for advanced prostate cancers.     

Prostate cancer risk in testosterone-treated men

Men with classical androgen deficiency have reduced prostate volume and blood prostate-specific antigen (PSA) levels compared with their age peers. As it is plausible that androgen deficiency partially protects against prostate disease, and that restoring androgen exposure increases risk to that of eugonadal men of the same age, men using ART should have age-appropriate surveillance for prostate disease. This should comprise rectal examination and blood PSA measurement at regular intervals (determined by age and family history) according to the recommendations, permanently revisited, published by ISSAM, EAU, Endocrine Society….

Testosterone replacement therapy is now being prescribed more often for aging men, the same population in which prostate cancer incidence increases; it has been suggested that administration in men with unrecognised prostate cancer might promote the development of clinically significant disease. In hypogonadal men who were candidates for testosterone therapy, a 14% incidence of occult cancer was found. A percentage (15.2%) of prostate cancer has been found in the placebo group (with normal DRE and PSA) in the prostate cancer prevention study investigating the chemoprevention potential of finasteride.

The hypothesis that high levels of circulating androgens is a risk factor for prostate cancer is supported by the dramatic regression, after castration, of tumour symptoms in men with advanced prostate cancer. However these effects, seen at a very late stage of cancer development, may not be relevant to reflect the effects of variations within a physiological range at an earlier stage.

Data from all published prospective studies on circulating level of total and free testosterone do not support the hypothesis that high levels of circulating androgens are associated with an increased risk of prostate cancer. A study on a large prospective cohort of 10,049 men, contributes to the gathering evidence that the long standing “androgen hypothesis” of increasing risk with increasing androgen levels can be rejected, suggesting instead that high levels within the reference range of androgens, estrogens and adrenal androgens decrease aggressive prostate cancer risk. Indeed, high-grade prostate cancer has been associated with low plasma level of testosterone. Furthermore, pre-treatment total testosterone was an independent predictor of extraprostatic disease in patients with localized prostate cancer; as testosterone decreases, patients have an increased likelihood of non-organ confined disease and low serum testosterone levels are associated with positive surgical margins in radical retropubic prostatectomy.

A clinical implication of these results concerns androgen supplementation which has become easier to administer with the advent of transdermal preparations (patch or gel) that achieve physiological testosterone serum levels without supra physiological escape levels. During the clinical development of a new testosterone patch in more than 200 primary or secondary hypogonadal patients, no prostate cancer was diagnosed.     

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein–protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.