Metabolic reprogramming has been recognised as a hallmark in solid tumours. Malignant modification of the tumour’s bioenergetics provides energy for tumour growth and progression. Otto Warburg first reported these metabolic and biochemical changes in 1927. In prostate cancer (PCa) epithelial cells, the tumour metabolism also changes during development and progress. These alterations are partly driven by the androgen receptor, the key regulator in PCa development, progress, and survival. In contrast to other epithelial cells of different entities, glycolytic metabolism in prostate cells sustains physiological citrate secretion in the normal prostatic epithelium. In the early stages of PCa, citrate is utilised to power oxidative phosphorylation and fuel lipogenesis, enabling tumour growth and progression. In advanced and incurable castration-resistant PCa, a metabolic shift towards choline, amino acid, and glycolytic metabolism fueling tumour growth and progression has been described. Therefore, even if the metabolic changes are not fully understood, the altered metabolism during tumour progression may provide opportunities for novel therapeutic strategies, especially in advanced PCa stages. This review focuses on the main differences in PCa’s metabolism during tumourigenesis and progression highlighting glutamine’s role in PCa.

LIMK2 is a serine/threonine/tyrosine kinase that promotes tumor cell invasion and metastasis by phosphorylating cell proteins and altering their functions. There is a need to find tumor-specific substrates for LIMK2 in order to understand the downstream pathway of these substrates, their function, and how they are regulated by LIMK2. Recently, our labrotory identified LIMK2 as an excellent target for curing castration-resistant prostate cancer (CRPC). In this study, we identify two novel substrates for LIMK2 in CRPC: speckle-type POZ protein (SPOP), and Y-box binding protein-1 (YBX1). While LIMK2 negatively regulates SPOP, it positively regulates YBX1 − both by phosphorylation using in-vitro kinase assays. A study in our labrotory also proved that LIMK2 regulates Aurora A kinase (AURKA), where AURKA directly phosphorylates LIMK2. AURKA is a serine/threonine kinase that regulates cell cycle during mitosis; it is known to be upregulated, with uncontrolled activity, in many types of cancer, including prostate cancer. It is therefore important to identify new substrates for AURKA, especially in light of reported lethality in early embryonic mice, in association with AURKA-knockout. In other words, targeting AURKA directly may cause severe toxicity, a finding that has prevented direct inhibitors from passing Phase II clinical trials. In this study, we also identified SPOP and YBX1 as direct substrates for AURKA. Our results confirm what we know about the LIMK2/AURKA relationship: that AURKA negatively regulates SPOP and positively regulates YBX1. Targeting LIMK2 and AURKA indirectly through SPOP, YBX1 and its other substrates holds tremendous therapeutic potential in treating prostate cancer. With this, we open the door for researches to investigate the direct phosphorylation of SPOP and YBX1 in other types of cancer cells known to have overexpression in SPOP and/or YBX1.

No description available.

Prostate cancer is the most common cancers in men. Androgen receptor (AR) is the primary driver of prostate cancer progression, and androgens are the male hormones to activate AR to simulate prostate cancer cells to grow. Thus, current therapy focuses on stopping androgen biosynthesis or blocking AR. However, suppression of AR activity by androgen depletion or direct AR antagonist treatment leads to emergence of castrate-resistant prostate cancer. Sigma-2 receptor (σ2R) plays an important role in cholesterol homeostasis and has been implicated in carcinogenesis including prostate cancer. Its molecular identity remained elusive until 2017 when transmembrane protein 97 (TMEM97) was identified as the bona fide sigma-2 receptor. Past studies have shown that σ2R is highly expressed in various mammalian tumor cell lines and TMEM97 was proposed as an oncogene in various cancers. We hypothesized that TMEM97/σ2 receptor regulates the expression and activities of androgen receptor and its variants to promote prostate cancer proliferation, and it can be a novel target of intervention to reduce AR oncogenic signaling in prostate cancer. The goal of this study is to define the role of TMEM97/σ2 receptor in prostate cancer, to unveil the mechanisms of TMEM97-AR interactions, and to explore therapeutic opportunity targeting TMEM97/σ2 receptor.Here we demonstrated that elevated TMEM97/σ2 receptor was found in prostate tumors of higher malignancy and advanced stages, and was associated with reduced survival of prostate cancer patients. Increased expression of TMEM97 enhanced prostate cancer cell proliferation, clonogenicity, and enzalutamide resistance. Knockdown of σ2 receptor expression reduced prostate cancer cell growth and clonogenicity, and sensitized LNCaP and castration resistant 22Rv1 cells towards enzalutamide. There was a positive correlation between TMEM97 and AR expressions in prostate cancers. Sigma-2 receptor knockdown reduced the protein expression of AR and its AR-V7 variant under hormone deprivation condition. Decreased TMEM97 also downregulated AR activities by inhibiting expression of prostate cancer maker genes and AR target genes. Its physical interaction with sigma-1 receptor was noticed, which may contribute to the regulation of AR. Sigma-2 receptor agonist can stimulate AR target genes expressions while antagonist decreased them. In addition, sigma-2 receptor antagonist provided synergism with enzalutamide treatment, showing therapeutic potential to overcome the resistance to current AR-targeted therapies.

Docetaxel (DTX)-based regimen is the mainstay treatment against castration resistant prostate cancer (CRPC). However, significant side-effects of DTX mandate that strategies to chemosensitize CRPC cells be utilized. We investigated whether physiologically achievable concentrations of nelfinavir (NFR) and curcumin (CUR), known to target the endoplasmic reticulum (ER) stress and AKT pathways, can increase DTX cytotoxicity. A significant reduction (~70%) in survival of a CRPC cell line, C4-2B, was evident within 24 hrs post-exposure to a combination of DTX (10 nM), NFR (5 µM) and CUR (5 µM), as compared to DTX alone (~34%). This rapid cytotoxicity was not seen in non-tumorigenic RWPE-1 cells as well as in primary prostate epithelial cells (PrEC) and bone-marrow mesenchymal stem cells (BM-MSC). A significant increase in apoptosis was seen in C4-2B cells but not RWPE-1 cells, as indicated by DNA-fragmentation, caspase-3 assay, and PARP cleavage. A significant reduction in C4-2B-derived colony forming units (CFU) was observed following exposure to DTX-NFR-CUR combination (92%), as compared to DTX alone (34%). In C4-2B cells, immunodetection and real-time PCR studies showed that exposure to 3-drug combination drastically reduced AKT activation, increased unfolded protein response (UPR) markers, such as XBP-1 mRNA and phosphorylated eIF-2α, and increased ER-stress induced pro-apoptotic markers such as CHOP, ATF4 and TRIB3. In RWPE-1 cells, upregulation of CHOP was observed with DTX-NFR-CUR combination, but no increase in ATF-4 and TRIB3 were observed. In vivo studies using C4-2B tumor xenografts showed a significant reduction in tumor volume following 4 week exposure to the 3-drug combination, as compared to DTX alone. Immunohistochemistry (IHC) of tumor sections revealed decreased Ki-67 staining indicating reduced cell proliferation and increased TUNEL staining indicating apoptosis, in DTX-NFR-CUR treated mice as compared to DTX alone. Therefore, our studies show that NFR and CUR can provide a promising approach as an adjuvant therapy to chemosensitize CRPC to DTX therapy. / acase@tulane.edu

Le jugement pénal évolue sous l’influence de la création et du développement de procédures visant à accélérer les réponses à la commission des infractions. Un nouveau modèle procédural – le procès pénal accéléré – a progressivement vu le jour, dû à deux transformations majeures.La première de ces transformations résulte de la multiplication des figures du juge au travers de diverses délégations de la fonction de juger. De nombreux acteurs appartenant ou non à l’autorité judiciaire se voient conférer une fonction de juger traditionnellement dévolue au juge du siège. Certains appartiennent à l’autorité judiciaire comme le procureur de la République ou le juge unique. D’autres, extérieurs à l’autorité judiciaire – des professionnels non juges et des juges non professionnels – participent également au jugement pénal.La simplification de l’acte de juger constitue la seconde transformation. Le débat judiciaire se voit progressivement restreint à la faveur du recours au consentement de l’auteur de l’infraction et de l’évitement de l’audience contradictoire. Cette simplification est aussi rendue possible par l’encadrement des oppositions susceptibles d’être formées à l’encontre des procédures accélérées. Les droits de la défense du mis-en-cause sont ainsi circonscrits et l’action civile éventuelle de la victime est contournée afin de ne pas nuire à la célérité des procédures.La délégation de la fonction de juger et la simplification de l’acte de juger se combinent donc et façonnent, au-delà de l’hétérogénéité des dispositifs étudiés, ce nouveau modèle procédural. / The criminal trial has evolved under the influence of the creation and development of procedures aiming to accelerate response to offences committed. A new procedural model – the accelerated criminal trial – has progressively appeared, due to two main changes.The first change comes from the increase in the number of judging figures through various delegations of the judging function. Many protagonists, whether part of the judiciary or not, are given the judging function, traditionally assigned to a judge from the Bench. Some belong to the judiciary, such as the public prosecutor or the magistrate judging alone. Others, out of the judiciary – professionals who are not judges and judges who are not professionals – also take part in the criminal trial.The simplification of the judging process represents the second change. Closing submissions and summing-up are gradually limited thanks to the use of the consent of the offender and the avoidance of Court hearings in the presence of both parties. This simplification is also permitted by the restriction of potential challenges to accelerated procedures. The rights of defence thus suffer limitations and potential legal actions taken by victims are evaded not to be detrimental to the rapidity of procedures.Therefore, the delegation of the judging function combines with the simplification of the judging process to shape, beyond the heterogeneity of the examined procedures, this new procedural model.

The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate development including the androgen receptor. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression induces castrate resistant phenotype. The highly overexpressed AhR clone illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.

<p>  </p> <p><strong>ABSTRACT</strong></p> <p>The worldwide obesity prevalence has almost tripled since 1973 according to the World Health Organization. In the United States, the disease is especially prevalent, with a recorded prevalence of 41.9 percent in 2017, as reported by the National Health and Nutrition Examination Survey. Obesity is associated with an increased risk of heart disease, type 2 diabetes, stroke, non-alcoholic fatty liver disease (NAFLD), and certain types of cancers, including prostate cancer (PCa). While obesity is preventable and reversible, it is a relapsing disease that requires long-term intervention. Furthermore, accumulating evidence shows obesity is not simply a matter of lack of willpower but the re-wired and altered biology that may need medical treatment. Therefore, researchers have been searching for effective approaches to treat obesity and obesity-related diseases. To this end, my research focuses on exploring the role of the sterol O-acyltransferase (SOAT) enzyme and how the inhibition of the enzyme benefits the treatment of obesity and PCa. In addition, I also studied the molecular signatures of NAFLD, with a special focus on altered lipid metabolism using proteomics and determined the protein oligomerization profiles. The major lines of research are summarized in the following and discussed in greater detail in chapters 2 to 5. </p> <p>SOAT enzyme catalyzes the conversion of free cholesterol into its storage form, cholesteryl ester. Our group previously showed that increased SOAT1 expression is associated with increased adipogenesis <em>in vitro</em> and increased adiposity in adipose tissue. When SOAT1 activity was blocked using the pharmacological inhibitor avasimibe, lipid droplet formation and expansion during adipogenesis were suppressed. We further showed that non-orally administered avasimibe led to significant fat mass loss in diet-induced obese (DIO) mice with concomitant food intake suppression and decreased expression of lipogenic genes in adipose tissue. Based on the promising use of avasimibe as an anti-obesity medication, I sought to answer whether avasimibe can enhance the weight loss effect of glucagon-like peptide-1 receptor agonist (GLP-1RA) by accelerating fat mass loss (chapter 2). We found subcutaneous administration of avasimibe can significantly potentiate the weight-reducing effect of the GLP-1RA in DIO mice.</p> <p>Inspired by the lipid droplet modulatory role of the SOAT1 enzyme, I also expanded my dissertation project to cancer (chapter 3). I found that low SOAT1 expression is associated with favorable patient outcomes among PCa patients who had previously undergone anti-hormone therapy. Since the current treatment option, anti-androgen drug enzalutamide, induces mechanisms of resistance in a short period, I hypothesized that blockage of SOAT1 activity using avasimibe would enhance the enzalutamide action and help overcome the resistance. To test this hypothesis, I characterized lipidomic signatures of PCa cells in response to enzalutamide and avasimibe treatments. Then, I tested the anti-cancer effect of the combined treatment in cell cultures and in xenograft tumors in nude mice. I found the combined treatment was significantly more effective in inhibiting cancer cell proliferation and tumor growth than each drug treatment alone. These findings provide insights into molecular signatures associated with enzalutamide treatment outcomes and can serve as a prelude to developing a therapeutic regimen targeting cholesterol metabolism. </p> <p>Among the comorbidities of obesity, NAFLD is very common in obese adults and the prevalence is close to 50–90%. We launched the third project (chapter 4) where we compared the liver proteome from lean mice and DIO mice. To date, most of the omics studies on DIO have been monolithic and very few have explored the multi-omic aspects of fatty liver tissue. To address this gap, we integrated global proteomics, phosphoproteomics and lipidomics to determine molecular signatures of the fatty liver. We identified a range of biological pathways that were altered, and we showed how the alterations in lipid content and amount were correlated with the alterations in the liver proteome and phosphoproteome. The results shed light on the interrelated nature of these biological processes. This was hypothesis-generating study that provided extensive data that could guide future investigations. </p> <p>We followed up on the third project and employed extensive measures to determine the protein oligomerization profiles of the fatty liver (chapter 5). Understanding the modes of protein oligomerization is important since proteins typically exert their biological functions by interacting with other proteins to form protein complexes. We used size exclusion chromatography (SEC) to fractionate liver proteins into 32 fractions based on their size and conducted label-free quantitation of each fraction using mass spectrometry. We successfully obtained elution profiles of individual proteins for subsequent comparison. Our approach enabled the identification of 1172 proteins found common in four liver samples (two lean livers and two fatty livers) for correlation profiling. We discovered that protein elution profiles were highly conserved in the fatty livers despite the metabolic disease state. At the same time, we identified several proteins with different elution profiles between the lean and the fatty livers, which could potentially mediate the hepatic dysfunctions displayed in NAFLD. This study delivers novel pieces of information about protein complex formation in fatty livers.</p> <p>The four research projects included in this dissertation explored obesity and obesity related diseases. Cholesterol accumulation is manifested as lipid metabolism is altered during the progression of obesity in adipose tissue and in PCa. Pharmacological inhibition of SOAT is responsible for cholesterol accumulation was effective in weight management in DIO and demonstrated anti-cancer effect in PCa models together with enzalutamide. These findings suggest that SOAT could be a therapeutic target for diseases featuring cholesteryl ester accumulation. Subsequent projects explored the liver proteome and revealed. In the subsequent projects, liver proteome showed a clear distinction between lean mice and obese mice. The identified proteins in these studies could facilitate the development of targeted therapies for treating NAFLD.</p>

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Yes / The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide. / University of Turin (Ricerca Locale grant 2014 and 2015) and Prostate Cancer UK grant S12-027