Bovine Lactoferrin Induces Cell Death in Human Prostate Cancer Cells.

Bovine lactoferrin (bLf) is a multifunctional protein widely associated with anticancer activity. Prostate cancer is the second most frequent type of cancer worldwide. This study was aimed at evaluating the influence of bLf on cell viability, cell cycle progression, reactive oxygen species (ROS) production, and rate of apoptosis in the human prostate cancer cell line (DU-145). MTT assay and trypan blue exclusion were used to analyze cell viability. Morphological changes were analyzed through optical microscopy after 24 h and 48 h of bLf treatment. FITC-bLf internalization and cellular damage were observed within 24 h by confocal fluorescence microscopy. Cell cycle analyses were performed by flow cytometry and propidium iodide. For caspases 3/7 activation and reactive oxygen species production evaluation, cells were live-imaged using the high-throughput system Operetta. The cell viability assays demonstrated that bLf induces cell death and morphological changes after 24 h and 48 h of treatment compared to control on DU-145 cells. The bLf internalization was detected in DU-145 cells, G1-phase arrest of the cell cycle, caspase 3/7 activation, and increased oxidative stress on bLf-treated cells. Our data support that bLf has an important anticancer activity, thus offering new perspectives in preventing and treating prostate cancer.

A protein coupling and molecular simulation analysis of the clinical mutants of androgen receptor revealed a higher binding for Leupaxin, to increase the prostate cancer invasion and motility.

Recently a novel coactivator, Leupaxin (LPXN), has been reported to interact with Androgen receptor (AR) and play a significant role in the invasion and progression of prostate cancer. The interaction between AR and LPXN occurs in a ligand-dependent manner and has been reported that the LIM domain in the Leupaxin interacts with the LDB (ligand-binding domain) domain AR. However, no detailed study is available on how the LPXN interacts with AR and increases the (prostate cancer) PCa progression. Considering the importance of the novel co-activator, LPXN, the current study also uses state-of-the-art methods to provide atomic-level insights into the binding of AR and LPXN and the impact of the most frequent clinical mutations H874Y, T877A, and T877S on the binding and function of LPXN. Protein coupling analysis revealed that the three mutants favour the robust binding of LPXN than the wild type by altering the hydrogen bonding network. Further understanding of the binding variations was explored through dissociation constant prediction which demonstrated similar reports as the docking results. A molecular simulation approaches further revealed the dynamic features which reported variations in the dynamics stability, protein packing, hydrogen bonding network, and residues flexibility index. Furthermore, we also assessed the protein motion and free energy landscape which also demonstrated variations in the internal dynamics. The binding free energy calculation revealed -32.95 ± 0.17 kcal/mol for the wild type, for H874Y the total binding energy (BFE) was -36.69 ± 0.11 kcal/mol, for T877A the BFE was calculated to be -38.78 ± 0.17 kcal/mol while for T877S the BFE -41.16 ± 0.12 kcal/mol. This shows that the binding of LPXN is increased by these mutations which consequently increase the PCa invasion and motility. In conclusion, the current study helps in understanding the protein networks and particular the coupling of AR-LPXN in prostate cancer and is of great interest in deciphering the molecular mechanism of disease and therapeutics developments.

Mathematical description of the effect of HIF inhibition on the radiobiological response of LNCaP cells.

According to the National Institute of Public Health, prostate cancer (PCa) is the leading cause of cancer death in Mexican men, highly associated with aggressiveness, resistance to treatment, and metastatic spread (Bharti et al., 2019) mediated by activation of the hypoxia-inducible factor 1 (HIF-1α). The objective of the present study was to evaluate the participation of HIF-1α activation in the radiobiological response of the human prostate adenocarcinoma cell line LNCaP, describing the phenomena with a mathematical model. Four groups were formed under different exposure conditions, including hypoxic cells treated with CoCl2 (300 µM for 22 h) with or without hypoxia-inducible factor inhibitor (150 nM chetomin for 4 h added after an incubation period of 18 h with CoCl2, just before completing the incubation period of 22 h). They were exposed to a source of 60Co in a dose range between 2 and 10 Gy to obtain survival curves that are fitted to a mathematical model. CoCl2 or chetomin treatments do not affect the viability of LNCaP cells that remained unchanged after irradiation. CoCl2 induced hypoxia reduces the survivability of LNCaP, and obstruction of HIF-1α signaling with chetomine produces a slight radioprotective effect. As others report, the genetic reprogramming induced by HIF-1α activation acts as an intrinsic agent that selects cells with more aggressive behavior (Pressley et al., 2017), while chetomin protects cells from death due to its scavenger properties. Interestingly, treatment with chetomin of cells induced to hypoxia (HIF-1 activation with CoCl2) produces a significant reduction in the radioresistance of LNCaP cells, demonstrating that the simultaneous use of chetomin and gamma radiation is an effective option for the treatment of hypoxic prostate cancer. At the molecular level, we suggest that the selective force exerted by HIF-1α depends on the production of free radicals by radiation. The proposed mathematical model showed that the rate of change in cell survival as a function of radiation dose is proportional to the product of two functions, one that describes cell death and the other that describes natural or artificial resistance to radiation.

Novel Dormancy Mechanism of Castration Resistance in Bone Metastatic Prostate Cancer Organoids.

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.

Design, synthesis, and characterization of PROTACs targeting the androgen receptor in prostate and lung cancer models.

Although the androgen receptor (AR) is a validated target for the treatment of prostate cancer, resistance to antiandrogens necessitates the development of new therapeutic modalities. Exploiting the ubiquitin-proteasome system with proteolysis-targeting chimeras (PROTACs) has become a practical approach to degrade specific proteins and thus to extend the portfolio of small molecules used for the treatment of a broader spectrum of diseases. Herein, we present three subgroups of enzalutamide-based PROTACs in which only the exit vector was modified. By recruiting cereblon, we were able to demonstrate the potent degradation of AR in lung cancer cells. Furthermore, the initial evaluation enabled the design of an optimized PROTAC with a rigid linker that degraded AR with a DC50 value in the nanomolar range. These results provide novel AR-directed PROTACs and a clear rationale for further investigating AR involvement in lung cancer models.

The secret identities of TMPRSS2: Fertility factor, virus trafficker, inflammation moderator, prostate protector and tumor suppressor.

The human TMPRSS2 gene is pathogenetically implicated in both coronaviral lung infection and prostate cancer, suggesting its potential as a drug target in both contexts. SARS-COV-2 spike polypeptides are primed by the host transmembrane TMPRSS2 protease, triggering virus fusion with epithelial cell membranes followed by an endocytotic internalisation process that bypasses normal endosomal activation of cathepsin-mediated innate immunity; viral co-opting of TMPRSS2 thus favors microbial survivability by attenuating host inflammatory responses. In contrast, most early hormone-dependent prostate cancers express TMPRSS2:ERG fusion genes arising from deletions that eliminate the TMPRSS2 coding region while juxtaposing its androgen-inducible promoter and the open reading frame of ERG, upregulating pro-inflammatory ERG while functionally disabling TMPRSS2. Moreover, inflammatory oxidative DNA damage selects for TMPRSS2:ERG-fused cancers, whereas patients treated with antiinflammatory drugs develop fewer of these fusion-dependent tumors. These findings imply that TMPRSS2 protects the prostate by enabling endosomal bypass of pathogens which could otherwise trigger inflammation-induced DNA damage that predisposes to TMPRSS2:ERG fusions. Hence, the high oncogenic selectability of TMPRSS2:ERG fusions may reflect a unique pro-inflammatory synergy between androgenic ERG gain-of-function and fusogenic TMPRSS2 loss-of-function, cautioning against the use of TMPRSS2-inhibitory drugs to prevent or treat early prostate cancer.

Prostate adenocarcinoma and COVID-19: The possible impacts of TMPRSS2 expressions in susceptibility to SARS-CoV-2.

TMPRSS2 (OMIM: 602060) is a cellular protease involved in many physiological and pathological processes, and it facilitates entry of viruses such as SARS-CoV-2 into host cells. It is important to predict the prostate's susceptibility to SARS-CoV-2 infection in cancer patients and the disease outcome by assessing TMPRSS2 expression in cancer tissues. In this study, we conducted the expression profiles of the TMPRSS2 gene for COVID-19 in different normal tissues and PRAD (prostate adenocarcinoma) tumour tissues. TMPRSS2 is highly expressed in normal tissues including the small intestine, prostate, pancreas, salivary gland, colon, stomach, seminal vesicle and lung, and is increased in PRAD tissues, indicating that SARS-CoV-2 might attack not only the lungs and other normal organs, but also in PRAD cancer tissues. Hypomethylation of TMPRSS2 promoter may not be the mechanism for TMPRSS2 overexpression in PRAD tissues and PRAD pathogenesis. TMPRSS2 expresses eleven isoforms in PRAD tissues, with the TMPRSS2-001 isoform expressed highest and followed by TMPRSS2-201. Further isoform structures prediction showed that these two highly expressed isoforms have both SRCR_2 and Trypsin (Tryp_SPc) domains, which may be essential for TMPRSS2 functional roles for tumorigenesis and entry for SARS-CoV-2 in PRAD patients. Analyses of functional annotation and enrichment in TMPRSS2 showed that TMPRSS2 is mostly enriched in regulation of viral entry into host cells, protein processing and serine-type peptidase activity. TMPRSS2 is also associated with prostate gland cancer cell expression, different complex(es) formation, human influenza and carcinoma, pathways in prostate cancer, influenza A, and transcriptional misregulation in cancer. Altogether, even though high expression of TMPRSS2 may not be favourable for PRAD patient's survival, increased expression in these patients should play roles in susceptibility of the SARS-CoV-2 infection and clinical severity for COVID-19, highlighting the value of protective actions of PRAD cases by targeting or androgen-mediated therapeutic strategies in the COVID-19 pandemic.

Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a second-generation antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and Ad-ACE2-transduced mice. Tmprss2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cells, however, such antiviral efficacy was lacking in human lung cells and organoids. Accordingly, enzalutamide showed no antiviral activity due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 regulatory locus in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19 through reducing TMPRSS2 expression in lung cells.

Estrogen and Androgen Receptor Inhibitors: Unexpected Allies in the Fight Against COVID-19.

TRANSLATIONAL RELEVANCE: No prophylactic treatments for COVID-19 have been clearly proven and found. In this pandemic context, cancer patients constitute a particularly fragile population that would benefit the best from such treatments, a present unmet need. TMPRSS2 is essential for COVID-19 replication cycle and it is under androgen control. Estrogen and androgen receptor dependent cues converge on TMPRSS2 regulation through different mechanisms of action that can be blocked by the use of hormonal therapies. We believe that there is enough body of evidence to foresee a prophylactic use of hormonal therapies against COVID-19 and this hypothesis can be easily tested on cohorts of breast and prostate cancer patients who follow those regimens. In case of pandemic, if the protective effect of hormonal therapies will be proven on cancer patients, the use of specific hormonal therapies could be extended to other oncological groups and to healthy individuals to decrease the overall risk of infection by SARS-CoV-2.Given the COVID-19 coronavirus emergency, a special focus is needed on the impact of this rapidly spreading viral infection on cancer patients. Androgen receptor (AR) signaling in the transmembrane protease serine 2 (TMPRSS2) regulation is emerging as an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility. In our study, we analyzed AR and TMPRSS2 expression in 17,352 normal and 9,556 cancer tissues from public repositories and stratified data according to sex and age. The emerging picture is that some patient groups may be particularly susceptible to SARS-CoV-2 infection and may benefit from antiandrogen- or tamoxifen-based therapies. These findings are relevant to choose proper treatments in order to protect cancer patients from concomitant SARS-CoV-2 contagion and related symptoms and put forward the idea that hormonal therapies could be used as prophylactic agents against COVID-19.

Sex differences in SARS-CoV-2 infection rates and the potential link to prostate cancer.

The recent outbreak of infections and the pandemic caused by SARS-CoV-2 represent one of the most severe threats to human health in more than a century. Emerging data from the United States and elsewhere suggest that the disease is more severe in men. Knowledge gained, and lessons learned, from studies of the biological interactions and molecular links that may explain the reasons for the greater severity of disease in men, and specifically in the age group at risk for prostate cancer, will lead to better management of COVID-19 in prostate cancer patients. Such information will be indispensable in the current and post-pandemic scenarios.