Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia.

In our previous studies, we showed that the generation of ovarian tumors in NSG mice (immune-compromised) resulted in the induction of muscle and cardiac cachexia, and treatment with withaferin A (WFA; a steroidal lactone) attenuated both muscle and cardiac cachexia. However, our studies could not address if these restorations by WFA were mediated by its anti-tumorigenic properties that might, in turn, reduce the tumor burden or WFA's direct, inherent anti-cachectic properties. To address this important issue, in our present study, we used a cachectic model induced by the continuous infusion of Ang II by implanting osmotic pumps in immunocompetent C57BL/6 mice. The continuous infusion of Ang II resulted in the loss of the normal functions of the left ventricle (LV) (both systolic and diastolic), including a significant reduction in fractional shortening, an increase in heart weight and LV wall thickness, and the development of cardiac hypertrophy. The infusion of Ang II also resulted in the development of cardiac fibrosis, and significant increases in the expression levels of genes (ANP, BNP, and MHCß) associated with cardiac hypertrophy and the chemical staining of the collagen abundance as an indication of fibrosis. In addition, Ang II caused a significant increase in expression levels of inflammatory cytokines (IL-6, IL-17, MIP-2, and IFNγ), NLRP3 inflammasomes, AT1 receptor, and a decrease in AT2 receptor. Treatment with WFA rescued the LV functions and heart hypertrophy and fibrosis. Our results demonstrated, for the first time, that, while WFA has anti-tumorigenic properties, it also ameliorates the cardiac dysfunction induced by Ang II, suggesting that it could be an anticachectic agent that induces direct effects on cardiac muscles.

Overview of follicle stimulating hormone and its receptors in reproduction and in stem cells and cancer stem cells.

Follicle stimulating hormone (FSH) and its receptor (FSHR) have been reported to be responsible for several physiological functions and cancers. The responsiveness of stem cells and cancer stem cells towards the FSH-FSHR system make the function of FSH and its receptors more interesting in the context of cancer biology. This review is comprised of comprehensive information on FSH-FSHR signaling in normal physiology, gonadal stem cells, cancer cells, and potential options of utilizing FSH-FSHR system as an anti-cancer therapeutic target.

Withaferin A and Ovarian Cancer Antagonistically Regulate Skeletal Muscle Mass.

Cachexia is a complex wasting syndrome that overwhelmingly affects the majority of late-stage cancer patients. Additionally, there are currently no efficacious therapeutic agents to treat the muscle atrophy induced by the cancer. While several preclinical studies have investigated the molecular signals orchestrating cachexia, very little information exists pertaining to ovarian cancer and the associated cachexia. Work from our lab has recently demonstrated that the steroidal lactone Withaferin A (WFA) is capable of attenuating the atrophying effects of ovarian cancer in a preclinical mouse model. However, it remained to be determined whether WFA's effect was in response to its anti-tumorigenic properties, or if it was capable of targeting skeletal muscle directly. The purpose of this study was to uncover whether WFA was capable of regulating muscle mass under tumor-free and tumor-bearing conditions. Treatment with WFA led to an improvement in functional muscle strength and mass under tumor-bearing and naïve conditions. WFA and ovarian cancer were observed to act antagonistically upon critical skeletal muscle regulatory systems, notably myogenic progenitors and proteolytic degradation pathways. Our results demonstrated for the first time that, while WFA has anti-tumorigenic properties, it also exerts hypertrophying effects on skeletal muscle mass, suggesting that it could be an anti-cachectic agent in the settings of ovarian cancer.

Withaferin A: a potential therapeutic agent against COVID-19 infection.

The outbreak and continued spread of the novel coronavirus disease 2019 (COVID-19) is a preeminent global health threat that has resulted in the infection of over 11.5 million people worldwide. In addition, the pandemic has claimed the lives of over 530,000 people worldwide. Age and the presence of underlying comorbid conditions have been found to be key determinants of patient mortality. One such comorbidity is the presence of an oncological malignancy, with cancer patients exhibiting an approximate two-fold increase in mortality rate. Due to a lack of data, no consensus has been reached about the best practices for the diagnosis and treatment of cancer patients. Interestingly, two independent research groups have discovered that Withaferin A (WFA), a steroidal lactone with anti-inflammatory and anti-tumorigenic properties, may bind to the viral spike (S-) protein of SARS-CoV-2. Further, preliminary data from our research group has demonstrated that WFA does not alter expression of ACE2 in the lungs of tumor-bearing female mice. Downregulation of ACE2 has recently been demonstrated to increase the severity of COVID-19. Therefore, WFA demonstrates real potential as a therapeutic agent to treat or prevent the spread of COVID-19 due to the reported interference in viral S-protein to host receptor binding and its lack of effect on ACE2 expression in the lungs.

Withaferin A attenuates ovarian cancer-induced cardiac cachexia.

Cachexia is a common multifactorial syndrome in the advanced stages of cancer and accounts for approximately 20-30% of all cancer-related fatalities. In addition to the progressive loss of skeletal muscle mass, cancer results in impairments in cardiac function. We recently demonstrated that WFA attenuates the cachectic skeletal muscle phenotype induced by ovarian cancer. The purpose of this study was to investigate whether ovarian cancer induces cardiac cachexia, the possible pathway involved, and whether WFA attenuates cardiac cachexia. Xenografting of ovarian cancer induced cardiac cachexia, leading to the loss of normal heart functions. Treatment with WFA rescued the heart weight. Further, ovarian cancer induced systolic dysfunction and diastolic dysfunction Treatment with WFA preserved systolic function in tumor-bearing mice, but diastolic dysfunction was partially improved. In addition, WFA abrogated the ovarian cancer-induced reduction in cardiomyocyte cross-sectional area. Finally, treatment with WFA ameliorated fibrotic deposition in the hearts of tumor-bearing animals. We observed a tumor-induced MHC isoform switching from the adult MHCα to the embryonic MHCß isoform, which was prevented by WFA treatment. Circulating Ang II level was increased significantly in the tumor-bearing, which was lowered by WFA treatment. Our results clearly demonstrated the induction of cardiac cachexia in response to ovarian tumors in female NSG mice. Further, we observed induction of proinflammatory markers through the AT1R pathway, which was ameliorated by WFA, in addition to amelioration of the cachectic phenotype, suggesting WFA as a potential therapeutic agent for cardiac cachexia in oncological paradigms.

Withaferin A ameliorates ovarian cancer-induced cachexia and proinflammatory signaling.

BACKGROUND: Ovarian cancer is the fifth leading cause of cancer-related deaths amongst women in the United States. Cachexia is the primary cause of death in approximately 30% of cancer patients, and is often evidenced in ovarian cancer patients. We tested the steroidal lactone Withaferin A to examine if it could ameliorate ovarian cancer-induced cachexia. METHODS: Six-week-old severely immunodeficient female mice were xenografted with the ovarian cancer cell line A2780 followed by treatment with Withaferin A or vehicle. Changes in functional grip strength were assessed on a weekly basis. Postmortem, H&E staining was performed on skeletal muscle sections and immunofluorescent immunohistochemistry was performed on skeletal muscle and tumor sections. The levels of NF-κB-related proinflammatory cytokines were assessed in the xenografted tumors and in resident host skeletal muscle. RESULTS: Xenografting of the A2780 cell line resulted in a significant rate of mortality, which was attenuated by a therapeutic dosage of Withaferin A. Mice that received vehicle treatment following xenografting exhibited functional muscle decline over the course of the study. The therapeutic dosage Withaferin A treatment attenuated this reduction in grip strength, whereas the supratherapeutic dosage of Withaferin A was found to be toxic/lethal and demonstrated a further decline in functional muscle strength and an increased rate of mortality on par with vehicle treatment. At a histological level, the vehicle treated tumor-bearing mice exhibited a profound reduction in myofibrillar cross-sectional area compared to the vehicle treated tumor-free control group. The atrophic changes induced by the xenografted tumor were significantly ameliorated by treatment with Withaferin A. The combination of functional muscle weakening and induction of myofibrillar atrophy corroborate a cachectic phenotype, which was functionally rescued by Withaferin A. Further, treatment completely abolished the slow-to-fast myofiber type conversion observed in the settings of cancer-induced cachexia. In both host resident skeletal muscle and the xenografted tumors, we report an increase in NF-κB-related proinflammatory cytokines that was reversed by Withaferin A treatment. Finally, we demonstrated that Withaferin A significantly downregulates cytosolic and nuclear levels of phospho-p65, the active canonical NF-κB transcription factor, in xenografted tumors. CONCLUSIONS: Cumulatively, our results demonstrate a previously overlooked role of Withaferin A in a xenograft model of ovarian cancer. We propose mechanisms by which Withaferin A reduces NF-κB-dependent pro-inflammatory cytokine production leading to an attenuation of the cachectic phenotype in an i.p. xenograft model of ovarian cancer.

PTTG1: a Unique Regulator of Stem/Cancer Stem Cells in the Ovary and Ovarian Cancer.

Origin of cancer stem cells (CSCs) and mechanisms by which oncogene PTTG1 contributes to tumor progression via CSCs is not known. Ovarian CSCs exhibit characteristics of self-renewal, tumor-initiation, growth, differentiation, drug resistance, and tumor relapse. A common location of putative origin, namely the ovarian surface epithelium, is shared between the normal stem and CSC compartments. Existence of ovarian stem cells and their co-expression with CSC signatures suggests a strong correlation between origin of epithelial cancer and CSCs. We hereby explored a putative oncogene PTTG1 (Securin), reported to be overexpressed in various tumors, including ovarian. We report a previously overlooked role of PTTG1 as a marker of CSCs thereby modulating CSC, germline, and stemness-related genes. We further characterized PTTG1's ability to regulate (cancer) stem cell-associated self-renewal and epithelial-mesenchymal transition pathways. Collectively, the data sheds light on a potential target expressed during ovarian tumorigenesis and metastatically disseminated ascites CSCs in the peritoneal cavity. Present study highlights this unconventional, under-explored role of PTTG1 in regulation of stem and CSC compartments in ovary, ovarian cancer and ascites and highlights it as a potential candidate for developing CSC specific targeted therapeutics.

Novel view of the adult stem cell compartment - a developmental story of germline and parental imprinting.

Evidence has accumulated that postnatal tissues contain developmentally early stem cells that remain in a dormant state as well as stem cells that are more proliferative, supplying tissue-specific progenitor cells and thus playing a more active role in the turnover of adult tissues. The most primitive, dormant, postnatal tissue-derived stem cells, called very small embryonic like stem cells (VSELs), are regulated by epigenetic changes in the expression of certain parentally imprinted genes, a molecular phenomenon previously described for maintaining primordial germ cells (PGCs) in a quiescent state. Specifically, they show erasure of parental imprinting at the Igf2-H19 locus, which keeps them in a quiescent state in a similar manner as migrating PGCs. To date, the presence of these cells in adult postnatal tissues have been demonstrated by at least 25 independent laboratories. We envision that similar changes in expression of parentally imprinted genes may also play a role in the quiescence of dormant VSELs present in other non-hematopoietic tissues. Recent data indicate that VSELs expand in vivo and in vitro after reestablishment of somatic imprinting at the Igf2-H19 locus by nicotinamide treatment in response to stimulation by pituitary gonadotrophins (follicle stimulating factor, luteinizing hormone and prolactin) and gonadal androgens and estrogens. These cells could be also successfully expanded ex vivo in the presence of the small molecule UM177.

PERK regulates skeletal muscle mass and contractile function in adult mice.

Skeletal muscle mass is regulated by the coordinated activation of several anabolic and catabolic pathways. The endoplasmic reticulum (ER) is a major site of protein folding and a reservoir for calcium ions. Accretion of misfolded proteins or depletion in calcium concentration causes stress in the ER, which leads to the activation of a signaling network known as the unfolded protein response (UPR). In the present study, we investigated the role of the protein kinase R-like endoplasmic reticulum kinase (PERK) arm of the UPR in the regulation of skeletal muscle mass and function in naive conditions and in a mouse model of cancer cachexia. Our results demonstrate that the targeted inducible deletion of PERK reduces skeletal muscle mass, strength, and force production during isometric contractions. Deletion of PERK also causes a slow-to-fast fiber type transition in skeletal muscle. Furthermore, short hairpin RNA-mediated knockdown or pharmacologic inhibition of PERK leads to atrophy in cultured myotubes. While increasing the rate of protein synthesis, the targeted deletion of PERK leads to the increased expression of components of the ubiquitin-proteasome system and autophagy in skeletal muscle. Ablation of PERK also increases the activation of calpains and deregulates the gene expression of the members of the FGF19 subfamily. Furthermore, the targeted deletion of PERK increases muscle wasting in Lewis lung carcinoma tumor-bearing mice. Our findings suggest that the PERK arm of the UPR is essential for the maintenance of skeletal muscle mass and function in adult mice.-Gallot, Y. S., Bohnert, K. R., Straughn, A. R., Xiong, G., Hindi, S. M., Kumar, A. PERK regulates skeletal muscle mass and contractile function in adult mice.

Canonical NF-κB signaling regulates satellite stem cell homeostasis and function during regenerative myogenesis.

Skeletal muscle regeneration in adults is attributed to the presence of satellite stem cells that proliferate, differentiate, and eventually fuse with injured myofibers. However, the signaling mechanisms that regulate satellite cell homeostasis and function remain less understood. While IKKß-mediated canonical NF-κB signaling has been implicated in the regulation of myogenesis and skeletal muscle mass, its role in the regulation of satellite cell function during muscle regeneration has not been fully elucidated. Here, we report that canonical NF-κB signaling is induced in skeletal muscle upon injury. Satellite cell-specific inducible ablation of IKKß attenuates skeletal muscle regeneration in adult mice. Targeted ablation of IKKß also reduces the number of satellite cells in injured skeletal muscle of adult mice, potentially through inhibiting their proliferation and survival. We also demonstrate that the inhibition of specific components of the canonical NF-κB pathway causes precocious differentiation of cultured satellite cells both ex vivo and in vitro. Finally, our results highlight that the constitutive activation of canonical NF-κB signaling in satellite cells also attenuates skeletal muscle regeneration following injury in adult mice. Collectively, our study demonstrates that the proper regulation of canonical NF-κB signaling is important for the regeneration of adult skeletal muscle.

MyD88 is required for satellite cell-mediated myofiber regeneration in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, leads to severe muscle wasting and eventual death of the afflicted individuals, primarily due to respiratory failure. Deficit in myofiber regeneration, potentially due to an exhaustion of satellite cells, is one of the major pathological features of DMD. Myeloid differentiation primary response 88 (MyD88) is an adaptor protein that mediates activation of various inflammatory pathways in response to signaling from Toll-like receptors and interleukin-1 receptor. MyD88 also regulates cellular survival, proliferation and differentiation in a cell-autonomous manner. However, the role of MyD88 in satellite stem cell homeostasis and function in dystrophic muscle remains unknown. In this study, we demonstrate that tamoxifen-inducible deletion of MyD88 in satellite cells causes loss of skeletal muscle mass and strength in the mdx mouse model of DMD. Satellite cell-specific deletion of MyD88 inhibits myofiber regeneration and stimulates fibrogenesis in dystrophic muscle of mdx mice. Deletion of MyD88 also reduces the number of satellite cells and inhibits their fusion with injured myofibers in dystrophic muscle of mdx mice. Ablation of MyD88 in satellite cells increases the markers of M2 macrophages without having any significant effect on M1 macrophages and expression of inflammatory cytokines. Finally, we found that satellite cell-specific deletion of MyD88 leads to aberrant activation of Notch and Wnt signaling in skeletal muscle of mdx mice. Collectively, our results demonstrate that MyD88-mediated signaling in satellite cells is essential for the regeneration of injured myofibers in dystrophic muscle of mdx mice.

MyD88 promotes myoblast fusion in a cell-autonomous manner.

Myoblast fusion is an indispensable step for skeletal muscle development, postnatal growth, and regeneration. Myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein that mediates Toll-like receptors and interleukin-1 receptor signaling. Here we report a cell-autonomous role of MyD88 in the regulation of myoblast fusion. MyD88 protein levels are increased during in vitro myogenesis and in conditions that promote skeletal muscle growth in vivo. Deletion of MyD88 impairs fusion of myoblasts without affecting their survival, proliferation, or differentiation. MyD88 regulates non-canonical NF-κB and canonical Wnt signaling during myogenesis and promotes skeletal muscle growth and overload-induced myofiber hypertrophy in mice. Ablation of MyD88 reduces myofiber size during muscle regeneration, whereas its overexpression promotes fusion of exogenous myoblasts to injured myofibers. Our study shows that MyD88 modulates myoblast fusion and suggests that augmenting its levels may be a therapeutic approach to improve skeletal muscle formation in degenerative muscle disorders.

Distinct roles of TRAF6 and TAK1 in the regulation of adipocyte survival, thermogenesis program, and high-fat diet-induced obesity.

Chronic low-grade inflammation, adipocyte hypertrophy, and glucose intolerance are common features of obesity and a risk factor for cancer. Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an adaptor protein that also possesses a non-conventional E3 ubiquitin ligase activity. In response to receptor-mediated events, TRAF6 activates transforming growth factor-activated kinase 1 (TAK1), which leads to activation of the MAPK and nuclear factor-kappa B (NF-κB) signaling pathways. However, the roles of TRAF6 and TAK1 in the regulation of adipocyte function remain less understood. Here, we demonstrate that adipocyte-specific deletion of TAK1, but not TRAF6, in mice reduces the survival of adipocytes and abundance of white adipose tissue (WAT). Adipocyte-specific ablation of TAK1, but not TRAF6, increases the expression for markers of beige/brown fat in WAT. Deletion of TAK1 in WAT increases phosphorylation of AMPK, abundance of PGC-1α, non-canonical NF-κB signaling, markers of M2 macrophages, and diminishes phosphorylation of JNK and canonical NF-κB signaling. Levels of TRAF6 and enzymatic activity of TAK1 are increased in WAT of mice fed with high-fat diet (HFD). Our results demonstrate that ablation of TAK1 drastically reduces HFD-induced obesity and improves energy expenditure and glucose metabolism. In contrast, adipocyte-specific ablation of TRAF6 has a minimal effect on HFD-induced obesity. Collectively, our results suggest that even though TRAF6 is an upstream activator of TAK1 in many signaling cascades, inactivation of TAK1, but not TRAF6, regulates adipocyte survival, energy expenditure, and HFD-induced obesity in mice.