Prostein expression on circulating tumor cells as a prognostic marker in metastatic castration-resistant prostate cancer.

BACKGROUND: Identification of emerging molecular biomarkers on circulating tumor cells (CTCs) represents an attractive feature of liquid biopsy that facilitates precision and tailored medicine in the management of metastatic castration-resistant prostate cancer (mCRPC). Prostein is an androgen-regulated transmembrane protein with high prostate specificity. Prostein-positive circulating tumor cell (CTC) was recently suggested to have diagnostic potential; however, no study has been conducted to evaluate its prognostic value in mCRPC. METHODS: CTCs from mCRPC patients were enumerated using the CellSearch System. Prostein-positive CTCs were identified by immunostaining results. The relationships between prostein expression on CTCs and PSA response rate, PSA progression-free survival (PSA-PFS), radiographic progression-free survival (PFS), and overall survival (OS) were tested by Fisher's exact test or evaluated using Kaplan-Meier and multivariate Cox analyses. RESULTS: Prostein-positive CTCs were identified in 31 of 87 baseline samples from mCRPC patients and 16 of 51 samples collected at the first follow-up visit. PSA response rates were significantly lower in baseline prostein-positive patients (0%, 0/31) than in prostein-negative patients (19.6%, 11/56) (p = 0.007). The 31 prostein-positive patients had significantly shorter PSA-PFS (p < 0.001), radiographic PFS (p < 0.001), and OS (p = 0.018), compared to the 56 prostein-negative patients at baseline. The association with PSA-PFS maintained its significance (p = 0.028) in multivariate analyses. Analyzing prostein expression at the first follow-up as well as the conversion of prostein expression from baseline to follow-up samples not only confirmed the association with PSA-PFS, but also demonstrated prognostic significance with OS. CONCLUSION: Our study provides the first evidence to support the potential of prostein expression on CTCs to serve as a novel prognostic marker in mCRPC patients. Future large-scale prospective studies are needed to validate our findings.

Sigma1 Regulates Lipid Droplet-mediated Redox Homeostasis Required for Prostate Cancer Proliferation.

Lipid droplets (LD) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer, LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. Prostate cancer arises as an androgen receptor (AR)-driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in prostate cancer cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and ROS pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive prostate cancer proliferation. SIGNIFICANCE: To proliferate, cancer cells must maintain productive metabolic and oxidative stress (eustress) while mitigating destructive, uncontrolled oxidative stress (distress). LDs are metabolic hubs that enable adaptive responses to promote eustress. Targeting the unique Sigma1 protein can trigger distress by disrupting the LD-mediated homeostasis required for proliferation.

Review of Cardiovascular Risk of Androgen Deprivation Therapy and the Influence of Race in Men with Prostate Cancer.

Androgen deprivation therapy is the cornerstone of prostate cancer therapy. Recent studies have revealed an association between androgen deprivation therapy and cardiovascular adverse effects such as myocardial infarction and stroke. This review summarizes the available research on the cardiovascular risk of men using androgen deprivation therapy. We also discuss racial disparities surrounding both prostate cancer and cardiovascular disease, emphasizing the importance of biological/molecular and socioeconomic factors in assessing baseline risk in patients beginning androgen ablation. Based on the literature, we provide recommendations for monitoring patients who are at high risk for a cardiovascular adverse event while being treated on androgen deprivation therapy. This review aims to present the current research on androgen deprivation therapy and cardiovascular toxicity with an emphasis on racial disparities and provides a framework for clinicians to decrease the cardiovascular morbidity in men that are being treated with hormone therapy.

Haloperidol and Prostate Cancer Prevention: More Epidemiologic Research Needed.

CONTEXT: The antipsychotic drug haloperidol has antiproliferative and growth-inhibiting properties on prostate cancer cell lines in vitro by binding the sigma 1 protein. Evidence is needed regarding a possible preventive association in men. OBJECTIVE: To examine whether our epidemiologic data support an inverse association of haloperidol use with risk of prostate cancer. DESIGN: These case-control analyses used conditional logistic regression to estimate relative risk by odds ratios (ORs) adjusting for race/ethnicity and aspects of medical care related to detection of prostate cancer. We tested 3 other commonly used antipsychotic drugs, risperidone, quetiapine, and olanzapine, for sigma 1 protein binding and inhibition of clonogenic growth of prostate cancer cells. Use of any of these by men was considered use of a comparator drug. MAIN OUTCOME MEASURES: 1) association of haloperidol with prostate cancer; 2) sigma 1 binding and clonogenic growth. RESULTS: Probably owing to small numbers of haloperidol recipients, evidence of a preventive association was inconsistent, depending on the definition of long-term use. If duration of use was greater than 1 year, the odds ratio (OR) was 0.38 (95% confidence interval (CI) = 0.14-1.01) for haloperidol and 0.80 (95% CI = 0.66-0.98) for the comparator drug; if the duration of use was greater than 2 years, the OR was 0.66 (95% CI = 0.24-1.76) for haloperidol and 0.84 (95% CI = 0.66-1.08) for the comparator drug. Unlike haloperidol, risperidone, quetiapine, and olanzapine did not bind sigma 1 or inhibit clonogenic growth. CONCLUSION: Given the laboratory evidence, our ambiguous epidemiologic findings should encourage more epidemiologic evaluation of haloperidol use and risk of prostate cancer. Finding a negative association could be a scientific advance in prostate cancer prevention but would not be sufficient basis for recommending the prescription of haloperidol for that purpose.

Small-Molecule Modulators of Sigma1 and Sigma2/TMEM97 in the Context of Cancer: Foundational Concepts and Emerging Themes.

There are two known subtypes of the so-called sigma receptors, Sigma1 and Sigma2. Sigma1 (encoded by the SIGMAR1 gene and also known as Sigma-1 receptor, S1R) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein that allosterically modulates the activity of its associated proteins. Sigma2, recently identified as transmembrane protein 97 (TMEM97), is an integral membrane protein implicated in cellular cholesterol homeostasis. A number of publications over the past two decades have suggested a role for both sigma proteins in tumor biology. Although there is currently no clinically used anti-cancer drug that targets Sigma1 or Sigma2/TMEM97, a growing body of evidence supports the potential of small-molecule compounds with affinity for these proteins, putative sigma ligands, as therapeutic agents to treat cancer. In preclinical models, these compounds have been reported to inhibit cancer cell proliferation, survival, adhesion, and migration; furthermore, they have been demonstrated to suppress tumor growth, to alleviate cancer-associated pain, and to exert immunomodulatory properties. Here, we will address the known knowns and the known unknowns of Sigma1 and Sigma2/TMEM97 ligand actions in the context of cancer. This review will highlight key discoveries and published evidence in support of a role for sigma proteins in cancer and will discuss several fundamental questions regarding the physiological roles of sigma proteins in cancer and sigma ligand mechanism of action.

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

Emerging evidence suggests that Sigma1 (SIGMAR1, also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy.Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR.

Introduction to Sigma Proteins: Evolution of the Concept of Sigma Receptors.

For over 40 years, scientists have endeavored to understand the so-called sigma receptors. During this time, the concept of sigma receptors has continuously and significantly evolved. With thousands of publications on the subject, these proteins have been implicated in various diseases, disorders, and physiological processes. Nevertheless, we are just beginning to understand what sigma proteins do and how they work. Two subtypes have been identified, Sigma1 and Sigma2. Whereas Sigma1 (also known as sigma-1 receptor, Sig1R, σ1 receptor, and several other names) was cloned over 20 years ago, Sigma2 (sigma-2 receptor, σ2 receptor) was cloned very recently and had remained a pharmacologically defined entity. In this volume, we will focus primarily on Sigma1. We will highlight several key subject areas in which Sigma1 has been well characterized as well as (re)emerging areas of interest. Despite the large number of publications regarding Sigma1, several fundamental questions remain unanswered or only partially answered. Most of what we know about Sigma1 comes from pharmacological studies; however, a clearly defined molecular mechanism of action remains elusive. One concept has become clear; Sigma1 is not a traditional receptor. Sigma1 is now considered a unique pharmacologically regulated integral membrane chaperone or scaffolding protein. A number of landmark discoveries over the past decade have begun to reshape the concept of sigma receptors. With the rapid emergence of new information, development of new tools, and changing conceptual frameworks, the field is poised for a period of accelerated progress.

Sigma1 Pharmacology in the Context of Cancer.

Sigma1 (also known as sigma-1 receptor, Sig1R, σ1 receptor) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein. The majority of publications on the subject have focused on the neuropharmacology of Sigma1. However, a number of publications have also suggested a role for Sigma1 in cancer. Although there is currently no clinically used anti-cancer drug that targets Sigma1, a growing body of evidence supports the potential of Sigma1 ligands as therapeutic agents to treat cancer. In preclinical models, compounds with affinity for Sigma1 have been reported to inhibit cancer cell proliferation and survival, cell adhesion and migration, tumor growth, to alleviate cancer-associated pain, and to have immunomodulatory properties. This review will highlight that although the literature supports a role for Sigma1 in cancer, several fundamental questions regarding drug mechanism of action and the physiological relevance of aberrant SIGMAR1 transcript and Sigma1 protein expression in certain cancers remain unanswered or only partially answered. However, emerging lines of evidence suggest that Sigma1 is a component of the cancer cell support machinery, that it facilitates protein interaction networks, that it allosterically modulates the activity of its associated proteins, and that Sigma1 is a selectively multifunctional drug target.

Novel small molecule guanidine Sigma1 inhibitors for advanced prostate cancer.

Prostate cancer is the most frequently diagnosed malignancy and the leading cause of cancer related death in men. First line therapy for disseminated disease relies on androgen deprivation, leveraging the addiction of these tumors on androgens for both growth and survival. Treatment typically involves antagonizing the androgen receptor (AR) or blocking the synthesis of androgens. Recurrence is common and within 2-3years patients develop castration resistant tumors that become unresponsive to AR-axis targeted therapies. In order to provide a more effective treatment, we are utilizing an approach that targets a key scaffolding protein, Sigma1 (also known as sigma-1 receptor), a unique 26-kilodalton integral membrane protein that is critical in stabilizing the AR. Herein we report on a new series of Sigma1 compounds for lead optimization derived from a hybrid pharmacophore approach.

Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer.

Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and ARv567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV-driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivoCancer Res; 77(9); 2439-52. ©2017 AACR.

Cyclopamine modulates γ-secretase-mediated cleavage of amyloid precursor protein by altering its subcellular trafficking and lysosomal degradation.

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-ß (Aß), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aß is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aß generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aß generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.

Sequential cytoprotective responses to Sigma1 ligand-induced endoplasmic reticulum stress.

The Sigma1 receptor (Sigma1) is an endoplasmic reticulum (ER) integral membrane protein that is highly expressed in a number of cancer cell lines. Small molecule compounds targeting Sigma1 (Sigma1 ligands) inhibit cancer cell proliferation and induce apoptotic cell death in vitro and inhibit tumor growth in xenograft experiments. However, the cellular pathways activated by Sigma1 protein-ligand interaction are not well defined. Here, we find that treatment with some Sigma1 ligands induces ER stress and activates the unfolded protein response (UPR) in a dose- and time-responsive manner in a range of adenocarcinoma cell lines. Autophagy is engaged after extended treatment with Sigma1 ligands, which suggests that protracted UPR results in autophagy as a secondary response. Inhibition of UPR by RNAi-mediated knockdown of inositol-requiring enzyme 1α and activating transcription factor 4 abrogates autophagosome formation, as does knockdown of essential autophagy gene products Beclin1 and autophagy protein 5. Knockdown of Sigma1 also suppresses IPAG [1-(4-iodophenyl)-3-(2-adamantyl) guanidine] induced UPR marker and autophagosome levels, indicating that this response is indeed Sigma1-mediated. We find that UPR activation precedes autophagosome formation and autophagy precedes apoptosis in Sigma1 ligand-treated cells. These processes are reversible, and washout of IPAG before cell death results in a return of autophagosomes and UPR markers toward basal levels. However, inhibition of Sigma1 ligand-induced UPR or autophagy accelerates apoptotic cell death. Together, these data suggest that UPR and autophagy are engaged as primary and secondary cytoprotective responses, respectively, to Sigma1 ligand-induced disruption of cancer cell protein homeostasis.

Inhibition of tumor cell growth by Sigma1 ligand mediated translational repression.

Treatment with sigma1 receptor (Sigma1) ligands can inhibit cell proliferation in vitro and tumor growth in vivo. However, the cellular pathways engaged in response to Sigma1 ligand treatment that contribute to these outcomes remain largely undefined. Here, we show that treatment with putative antagonists of Sigma1 decreases cell mass. This effect corresponds with repressed cap-dependent translation initiation in multiple breast and prostate cancer cell lines. Sigma1 antagonist treatment suppresses phosphorylation of translational regulator proteins p70S6K, S6, and 4E-BP1. RNAi-mediated knockdown of Sigma1 also results in translational repression, consistent with the effects of antagonist treatment. Sigma1 antagonist mediated translational repression and decreased cell size are both reversible. Together, these data reveal a role for Sigma1 in tumor cell protein synthesis, and demonstrate that small molecule Sigma1 ligands can be used as modulators of protein translation.

Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling.

Intrahost variations in the envelope receptor-binding domain (RBD) of HTLV-1 and STLV-1 primary isolates.

Four primate (PTLV), human (HTLV) and simian (STLV) T-cell leukemia virus types, have been characterized thus far, with evidence of a simian zoonotic origin for HTLV-1, HTLV-2 and HTLV-3 in Africa. The PTLV envelope glycoprotein surface component (SUgp46) comprises a receptor-binding domain (RBD) that alternates hypervariable and highly conserved sequences. To further delineate highly conserved motifs in PTLV RBDs, we investigated the intrahost variability of HTLV-1 and STLV-1 by generating and sequencing libraries of DNA fragments amplified within the RBD of the SUgp46 env gene. Using new and highly cross-reactive env primer pairs, we observed the presence of Env quasispecies in HTLV-1 infected individuals and STLV-1 naturally infected macaques, irrespective of the clinical status. These intrahost variants helped us to define highly conserved residues and motifs in the RBD. The new highly sensitive env PCR described here appears suitable for the screening of all known variants of the different PTLV types and should, therefore, be useful for the analysis of seroindeterminate samples.

HTLV-1 and -2 envelope SU subdomains and critical determinants in receptor binding.

BACKGROUND: Human T-cell leukemia virus (HTLV) -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU) of the HTLV envelope glycoprotein (Env) harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV) Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains. RESULTS: Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD) lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205), that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events. CONCLUSIONS: The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that contain all the determinants required for binding the HTLV receptor.

[Envelope capture by retroviruses]. / La capture d'enveloppe par les rétrovirus.

Retroelement transposition is a major source of diversity in genome evolution. Among the retrotransposable elements, the retroviruses are distinct in that their "transposition" extends from their initial host cells to neighboring cells and organisms. A determining step in the conversion of a retrotransposable element into an infectious retrovirus is the acquisition of an envelope glycoprotein, designated Env. Here, we review some examples of envelope "capture" by mammal retroviruses and provide evidence for such a mechanism by HTLV. This phenomenon may explain the notable conservation of env genes observed between phylogenetically distant retroviruses. Elucidation of these recombination processes should help to clarify retroviral phylogeny, better understand retroviral pathogenesis, and may lead to the identification of new retroelements.