Research & Initiatives
Androgen producing Stem Cells
Exogenous testosterone therapy can be used to treat testosterone deficiency; however, it has several adverse effects, including infertility, due to negative feedback on the hypothalamic-pituitary-gonadal (HPG) axis. Leydig stem cell (LSC) transplantation could provide a new strategy for treating testosterone deficiency, but the clinical translatability of injecting stem cells inside the testis is not feasible. We are exploring the feasibility of subcutaneously autografting LSCs in combination with Sertoli and myoid cells to increase testosterone. We are also studying whether the grafted LSCs can be regulated by the HPG axis and the molecular mechanism behind this regulation. For this research, we use LSCs isolated from the testes of C57BL/6 mice or human testis biopsies. These cells are grown in specialized culture conditions, which are supplemented with a growth factor cocktail that supports the growth and expansion of LSCs. Post characterization, these cells are subcutaneously grafted in combination with Sertoli cells and myoid cells into the animal models (for preclinical studies). Our results so far suggest that LSCs alone are incapable of self-renewal and differentiation. However, in combination with Sertoli cells and myoid cells, LSCs can undergo self-renewal as well as differentiation into mature Leydig cells. As a result, the recipient mice that received the LSC autograft showed testosterone production with preserved luteinizing hormone. Results also suggest that testosterone production from the graft is regulated by hedgehog (HH) signaling. These preliminary findings are the first to demonstrate that LSCs, when grafted subcutaneously in combination with Sertoli cells and myoid cells, can increase testosterone production. Therefore, highlighting LSC autograft as a potential new treatment for testosterone deficiency while simultaneously preserving the HPG axis.
Paracrine Factors from Testicular Microenviornment regulates Androgen production
Although testosterone deficiency (TD) may be present in one out of five men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). It is hypothesized that the TME releases paracrine factors to modulate LSC differentiation. For this purpose, our research uses cells (Sertoli, PMCs, LSCs, and ALCs) from men undergoing testis biopsies for sperm retrieval. These cells are evaluated for the paracrine factors in the presence or absence of the TME (Sertoli and PMC). The results so far demonstrated that TME secretes leptin, which induces LSC differentiation and increases testosterone production. Leptin's effects on LSC differentiation and testosterone production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin binds to the leptin receptor on LSCs and induces DHH signaling to modulate LSC differentiation. Leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function does not affect leptin levels. These findings identify leptin as a key paracrine factor released by cells within the TME that modulates LSC differentiation and testosterone release from mature Leydig cells, a finding with important clinical implications for TD.
Nitroso-redox Imbalance can Negative Impact Androgen Production
The cause of age-related changes in testosterone remains unclear. We hypothesized that increased nitroso-redox imbalance with aging could affect testosterone production. In this study, we measured several markers of nitroso-redox imbalance (4-HNE, 3-NT, and NT) in serum of S-nitrosoglutathione reductase knockout (GSNOR KO) mice that have increased nitroso-redox imbalance and compared these to wild-type (WT) mice. We evaluated the impact of age-induced nitroso-redox imbalance on serum luteinizing hormone (LH) and testosterone (T) in WT young (<2 months), middle-aged (2-6 months), and aged (>12 months) mice. Finally, to elucidate the susceptibility of testes to nitroso-redox imbalance, we measured 4-HNE protein levels in the testes of WT and KO mice. Our findings so far have identified increased nitroso-redox imbalance, as evidenced by increased protein levels of 4-HNE in the serum of GSNOR KO mice compared with WT mice. We demonstrated that 4-HNE serum protein levels increase in WT mice with age but do not accumulate in the testes. We also found that T levels were similar in all age groups. We found that serum LH levels in aged and middle-aged mice are increased compared to young mice, consistent with the phenotype of subclinical hypogonadism. This suggests that: Increased serum 4-HNE and LH levels without changes in T with age suggest that nitroso-redox imbalance is associated with subclinical hypogonadism in aged mice. Recognizing the relationship and etiology of a poorly understood classification of hypogonadism could be a paradigm shift in how age-related testosterone change is diagnosed and treated.
Overcoming nitroso-redox imbalance by Nitric oxide treatment can target Prostate Cancer growth by reducing tumor microenvironment
Prostate cancer is the second most frequent cause of cancer-related deaths in men. Men with prostate cancer that has recurred after local therapy usually respond to androgen deprivation therapy (ADT); however, despite this treatment, most patients eventually experience progression of the disease within 2 years, a condition known as castration-resistant prostate cancer (CRPC). In trying to understand the causes of this androgen resistance that develops in CRPC, most research has focused directly on the splice variants of the androgen receptor (ARVs). However, the tumor microenvironment (TME) has been shown to play a major role in tumor progression. Yet, the response to therapy in other cancer types has been inadequately studied in CRPC. TME is comprised of a variety of cell types, including immune cells, fibroblasts, pericytes, and tumor-associated macrophages (TAMs). TAMs are recruited to tumors from diverse signaling molecules such as chemokines (CCL-2 and CCL-5) and cytokines (IL-34 and CSF-1). While the exact mechanism is unknown, TAMs have been reported to play a key role in the progression of prostate cancer through the secretion of cytokines, matrix metalloproteinases, and growth factors.
A key molecule in the regulation of TME interactions is the ubiquitous nitric oxide (NO). We have previously established the importance of NO in the cardiovascular and immune systems and in male secondary hypogonadism. Others have investigated the tumoricidal implications of NO in therapeutic resistance, cell survival, the proliferation of tumors, inhibition of tumor growth, and reduction in lung metastases in many cancer types. Several NO donors, including S-nitrosothiols, organic nitrates, and Metal-NO complexes, have shown impacts on cancer progression. S-nitrosothiols such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) have also shown promising effects as antineoplastic agents. However, these studies have only focused on certain aspects of NO, such as its role in both growth and antigrowth effects, cellular localization, endogenous expression of the androgen receptor (AR), and AR function inactivation by S-nitrosylation.
Accordingly, we tested the hypothesis that increased NO will lead to tumor suppression of CRPC through the tumor microenvironment. Our findings showed that S-nitrosoglutathione (GSNO), an NO donor, decreased the tumor burden in the murine model of CRPC by targeting tumors in a cell nonautonomous manner. GSNO inhibited both the abundance of anti-inflammatory (M2) macrophages and the expression of pERK, indicating that tumor-associated macrophage activity is influenced by NO. Additionally, GSNO decreased IL-34, indicating suppression of tumor-associated macrophage differentiation. Cytokine profiling of CRPC tumor grafts exposed to GSNO revealed a significant decrease in expression of G-CSF and M-CSF compared with grafts not exposed to GSNO. We verified the durability of NO on CRPC tumor suppression by using secondary xenograft murine models. This study validates the significance of NO in the inhibition of CRPC tumors through the tumor microenvironment (TME). These findings may facilitate the development of previously unidentified NO-based therapy for CRPC.
Nitric oxide S-nitrosylates CSF1R to augment the action of CSF1R inhibition against castration resistant prostate cancer
This limited efficacy of hormonal therapy is believed to result in part from the unique ability of Prostate cancer to evolve through as-yet-unclear mechanisms in the tumor microenvironment (TME) that promote immune escape. TME is comprised of a variety of cell types of which tumor-associated macrophages (TAMs)7 frequently make up a substantial proportion and consists of two opposing phenotypes, classically activated (M1-like) and alternatively activated (M2 like), which have been correlated with anti and protumoral functions and with patient survival8. M1/M2 dichotomy is modulated via colony-stimulating factor 1 (CSF1), which binds to the CSF1 receptor (CSF1R) to control the proliferation, differentiation, and survival of macrophages. Studies suggest that blocking CSF1R could delay tumor growth via TAM reduction. However, single agent CSF1R blockade showed underwhelming results in phase 2 and 3 trials. One of the reasons for the limited efficacy is the feedback mechanism induced by activated CSF1R (in tumors) by recruitment of immunosuppressive and pro-tumoral TAMs and cytokines that are conducive to immune suppression16 and are not effectively targeted by single agent CSF1R blockade. Therefore, suggesting the need to find ways to keep a check on TAMs and cytokines that derail the efficiency of CSF1R blockade therapy.
In this context, another relevant yet understudied aspect is Nitric oxide synthase (NOS). In mammals, three NOS isoenzymes are found. Of these, Neuronal and endothelial NOS (nNOS/NOS1 and eNOS/ NOS3, respectively) are constitutively expressed, produce Nitric Oxide (NO)(nM), and are regulated by Ca2+ binding to calmodulin. NO has been shown to be involved in the regulation of adaptive immune responses by modulating T-cell activation and differentiation, promoting T-cell receptor-mediated signaling from the immune synapse and M1/M2 macrophage polarization. The effect of NO is mechanistically imposed by the covalent attachment of a nitroso group to a cysteine thiol (Protein S-nitrosylation). NOSs of tumor cells, in contrast, synthesize superoxide and peroxynitrite, which results in reduced tetrahydrobiopterin: dihydrobiopterin ratio (BH4:BH2). The reduced BH4:BH2 ratio results in the uncoupling of NOSs and is observed in multiple cancer types. One major impact of NOS uncoupling is the reduction in NO levels and increase in oxidative stress. Both of these aid the pro-tumorigenic cytokines such as NFkB23, IFNγ24, and TNFα25 which could further induce CSF1 expression in various cell types of TME and are not effectively targeted by single agent CSF1R blockade. Together, these findings warrant to study of the impact of uncoupling of NOSs in conjunction with CSF1-CSF1R mediated M1/M2 dichotomy and TME in PCa, which is unknown.
In this study, we found that in high-grade PCa human specimens, eNOS is positively correlated with CSF1-CSF1R signaling and remains uncoupled. The uncoupling disables eNOS to generate sufficient Nitric oxide (NO) that is required for inducing effective S-nitrosylation of CSF1R molecule at specific cysteine sites (Cys 224, Cys 278, and Cys 830). Importantly, we found that S-nitrosylation of CSF1R molecule at Cys 224, Cys 278, and Cys 830 sites is necessary for effective inhibition of tumor-promoting cytokines (which are downstream of CSF1-CSF1R signaling) by CSF1R blockade. In this context, we studied if exogenous NO treatment could rescue the side effects of eNOS uncoupling. Results showed that exogenous NO treatment (using S-nitrosoglutathione (GSNO)) is effective in not only inducing S-Nitrosylation of CSF1R molecule, but it helps in rescuing the excess oxidation in tumor regions, reducing overall tumor burden, suppressing the tumor-promoting cytokines which are ineffectively suppressed by CSF1R blockade. Together these results postulated that NO therapy could act as an effective combinatorial partner with CSF1R blockade against CRPC. In this context, results demonstrated that exogenous NO treatment successfully augments the anti-tumor ability of CSF1Ri in murine models of CRPC. Importantly, the overall tumor reduction was most effective in NO-CSF1Ri therapy compared to NO or CSF1Ri mono therapies. Moreover, Immunophenotyping of tumor grafts showed that the NO-CSF1Ri combination significantly decreased the intratumoral percentage of anti-inflammatory macrophages, myeloid-derived progenitor cells and increased the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells respectively. Together, our study suggests that the NO-CSF1Ri combination has the potential to act as a therapeutic agent that restores control over TME and improves the outcomes of PCa patients.
Studying Prostate Cancer Progression using Advance Machine Learning Modeling
A large number of deaths from any cancer type, including PCa, occurs due to challenges with timely diagnoses and prognoses of the disease, which also increases the overall risk and cost of the treatment. In PCa, diagnosis starts with prostate-specific antigen (PSA) level detection, which is above the normal range, the patient is subjected to genomic testing such as - 4K scores, PCA3, or PHI test. Once/if the results of these genomic tests return positive, magnetic resonant imaging (MRI) is used to identify potential areas of PCa. The biopsies are extracted by the clinicians, further inspected by the board-certified pathologist, and are then sent for genomic testing to confirm the severity of the disease. Typically, a single worst area is selected for the test, leaving a large area from consideration. In case of false positive/negative outcomes, patients are subjected to the same steps making it financially cumbersome for the patients and increasing the time/accuracy of treatment. We are working to develop the machine learning pipelines that will allow us to 1) investigate the entire areas of the biopsies at a granular level before assigning a score and 2) predict the course of tumor progression/regression. For this, the pipelines utilize a multi-tiered approach which includes a) generating a digital map of the tissue architecture, b) overlaying this with the patient’s genomics, c) assigning sub-scores to the entire image, and computing the grade of cancer by generalizing the scores. We have submitted a provisional patent with some of the preliminary data from this application, with support from the University of Miami. We aim to refine this technology to 1) establish the efficiency of the developed AI model in a clinical setting (using data from patients enrolled in active surveillance trials, 2) map disease using advanced CNN tools, 3) create a user-friendly interface to enhance clinical use of the AI model. Successful completion of this study will yield a paradigm-shifting technology that will aid in clinical decision-making, improve patient outcomes through early detection and accurate prognostic assessment of PCa, and could potentially extend to other cancer settings.
