Drug inhibition of redox factor-1 restores hypoxia-driven changes in tuberous sclerosis complex 2 deficient cells

Simple Summary: Tuberous sclerosis complex (TSC) is a genetic disease where patients are predisposed to tumors and neurological complications. Current therapies for this disease are not fully curative. We aimed to explore novel drug targets and therapies that could further benefit TSC patients. This work uncovered a novel pathway that drives disease in TSC cell models involving redox factor-1 (Ref-1). Ref-1 is a protein that turns on several key transcription factors that collectively promote tumor growth and survival through direct redox signaling. Processes regulated by Ref-1 include angiogenesis, inflammation, and metabolic transformation. Therefore, this work reveals a new drug target, where inhibitors of Ref-1 could have an additional benefit compared to current drug therapies. Abstract: Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone

Drug inhibition of redox factor-1 restores hypoxic-driven changes in Tuberous Sclerosis Complex 2-deficient cells

Therapies with mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for Tuberous Sclerosis Complex (TSC) patients. Here we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses redox signaling activity that stimulates the transcriptional activity of STAT3, NF-B, and HIF-1 involved in inflammation, proliferation, angiogenesis and hypoxia, respectively. Here we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor, APX3330, was effective at blocking the hyperactivity of STAT3, NF-B, and HIF-1. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors, such as STAT3, NF-B and HIF-1, as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits to just using mTORC1 inhibitors alone

Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir−bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis

Defining the role of folliculin and its interacting partners

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterised by benign hair follicle tumours, pneumothorax and renal cancer. Folliculin (FLCN), the protein product of FLCN, is a poorly characterised tumour suppressor, currently linked to multiple cellular pathways. Autophagy is an evolutionarily conserved biological process that maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase, ULK1, is known to initiate autophagy, the underlying mechanisms are still being unravelled and new ULK1 substrates are being identified. In this study, FLCN is identified as a new ULK1 substrate that interacts with the autophagy machinery via GABARAP. FLCN/GABARAP interaction is shown to be modulated by the presence of FNIP1 and FNIP2, as well as ULK1-directed phosphorylation of FLCN. Furthermore, loss of FLCN impairs autophagy while re-expression rescues autophagy. FLCN was found to interact with several Rab small G proteins linked to autophagy, implicating FLCN in vesicular trafficking. Upon generation of FNIP1/2 knockdown cell lines it was revealed that FNIP2 is possibly involved in the transcriptional regulation of FLCN and is involved in FNIP1’s translation and/or stability. Moreover, FNIP1 could be involved in regulation of FNIP2’s transcription via a negative feedback mechanism. FNIP proteins were further implicated in autophagy when FNIP1 and FNIP2 gene expression were found to be significantly increased upon starvation in HK2 cells. Additionally, p62 and GABARAP protein levels demonstrated a significant increase in FNIP2 knockdown cell lines suggestive of impaired autophagy. This work substantially contributes to our understanding of FLCN by linking it directly to autophagy. Furthermore, FLCN and the FNIPs are shown to be involved in multiple protein interactions which could mean that FLCN and its interacting partners might have a more universal housekeeping role, which when lost leads to cancer

FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1