Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

Hypoxia Induced ER Stress Response as an Adaptive Mechanism in Cancer.

It is evident that regions within tumors are deprived of oxygen, which makes the microenvironment hypoxic. Cancer cells experiencing hypoxia undergo metabolic alterations and cytoprotective adaptive mechanisms to survive such stringent conditions. While such mechanisms provide potential therapeutic targets, the mechanisms by which hypoxia regulates adaptive responses-such as ER stress response, unfolded protein response (UPR), anti-oxidative responses, and autophagy-remain elusive. In this review, we summarize the complex interplay between hypoxia and the ER stress signaling pathways that are activated in the hypoxic microenvironment of the tumors.

Natural tyrosinase enzyme inhibitors: A path from melanin to melanoma and its reported pharmacological activities.

The skin containing melanin pigment acts as a protective barrier and counteracts the UVR and other environmental stressors to maintain or restore disrupted cutaneous homeostasis. The production of melanin pigment is dependent on tyrosine levels. L-tyrosine and L-dihydroxyphenylalanine (L-DOPA) can serve both as a substrates and intermediates of melanin synthetic pathway and as inducers and positive regulators of melanogenesis. The biosynthesis of melanin is stimulated upon exposure to UVR, which can also stimulate local production of hormonal factors, which can stimulate melanoma development by altering the chemical properties of eu- and pheomelanin. The process of melanogenesis can be altered by several pathways. One involves activation of POMC, with the production of POMC peptides including MSH and ACTH, which increase intracellular cAMP levels, which activates the MITF, and helps to stimulate tyrosinase (TYR) expression and activity. Defects in OCA1 to 4 affects melanogenic activity via posttranslational modifications resulting in proteasomal degradation and reducing pigmentation. Further, altering, the MITF factor, helps to regulate the expression of MRGE in melanoma, and helps to increase the TYR glycosylation in ER. CRH stimulates POMC peptides that regulate melanogenesis and also by itself can stimulate melanogenesis. The POMC, P53, ACTH, MSH, MC1R, MITF, and 6-BH4 are found to be important regulators for pigmentation. Melanogenesis can affect melanoma behaviour and inhibit immune responses. Therefore, we reviewed natural products that would alter melanin production. Our special focus was on targeting melanin synthesis and TYR enzyme activity to inhibit melanogenesis as an adjuvant therapy of melanotic melanoma. Furthermore, this review also outlines the current updated pharmacological studies targeting the TYR enzyme from natural sources and its consequential effects on melanin production.

Three-dimensional growth sensitizes breast cancer cells to treatment with ferroptosis-promoting drugs.

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.

Analysis of ER-Phagy in Cancer Drug Resistance.

The ability of the cancer cells to survive hostile environment depends on their cellular stress response mechanisms. These mechanisms also help them to develop resistance to chemotherapies. Autophagy and more specifically organelle specific autophagy is one such adaptive mechanism that promotes drug resistance in cancer cells. Endoplasmic reticulum-specific autophagy or ER-phagy has been more recently described to overcome ER-stress through the degradation of damaged ER. ER-resident proteins such as FAM134B act as ER-phagy receptors to specifically target damaged ER for degradation through autophagy. Moreover, we had recently deciphered that ER-phagy facilitates cancer cell survival during hypoxic stress and we predict that this process could play a critical role in the development of drug resistance in cancer cells. Therefore, here, we provide a lay description of how ER-phagy could be investigated biochemically by Western blot analysis and silencing ER-phagy receptor genes using small interfering RNAs (siRNA).

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress.

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Crosstalk Between ER Stress, Autophagy and Inflammation.

The endoplasmic reticulum (ER) is not only responsible for protein synthesis and folding but also plays a critical role in sensing cellular stress and maintaining cellular homeostasis. Upon sensing the accumulation of unfolded proteins due to perturbation in protein synthesis or folding, specific intracellular signaling pathways are activated, which are collectively termed as unfolded protein response (UPR). UPR expands the capacity of the protein folding machinery, decreases protein synthesis and enhances ER-associated protein degradation (ERAD) which degrades misfolded proteins through the proteasomes. More recent evidences suggest that UPR also amplifies cytokines-mediated inflammatory responses leading to pathogenesis of inflammatory diseases. UPR signaling also activates autophagy; a lysosome-dependent degradative pathwaythat has an extended capacity to degrade misfolded proteins and damaged ER. Thus, activation of autophagy limits inflammatory response and provides cyto-protection by attenuating ER-stress. Here we review the mechanisms that couple UPR, autophagy and cytokine-induced inflammation that can facilitate the development of novel therapeutic strategies to mitigate cellular stress and inflammation associated with various pathologies.

Caenorhabditis elegans as a possible model to screen anti-Alzheimer's therapeutics.

Alzheimer's disease (AD) is regarded as one of the significant health burdens, as the prevalence is raising worldwide and gradually reaching to epidemic proportions. Consequently, a number of scientific investigations have been initiated to derive therapeutics to combat AD with a concurrent advancement in pharmacological methods and experimental models. Whilst, the available experimental pharmacological approaches both in vivo and in vitro led to the development of AD therapeutics, the precise manner by which experimental models mimic either one or more biomarkers of human pathology of AD is gaining scientific attentions. Caenorhabditis elegans (C. elegans) has been regarded as an emerging model for various reasons, including its high similarities with the biomarkers of human AD. Our review supports the versatile nature of C. elegans and collates that it is a well-suited model to elucidate various molecular mechanisms by which AD therapeutics elicit their pharmacological effects. It is apparent that C. elegans is capable of establishing the pathological processes that links the endoplasmic reticulum and mitochondria dysfunctions in AD, exploring novel molecular cascades of AD pathogenesis and underpinning causal and consequential changes in the associated proteins and genes. In summary, C. elegans is a unique and feasible model for the screening of anti-Alzheimer's therapeutics and has the potential for further scientific exploration.

Pharmacological STING Activation Is a Potential Alternative to Overcome Drug-Resistance in Melanoma.

Melanoma is the most aggressive type of skin cancer and resistance to the conventional chemotherapy is the major cause for its poor prognosis. Metabolic perturbations leading to increased production of reactive oxygen species activate NRF2-dependent anti-oxidative responses to survive oxidative stress. This protective function of NRF2 is the primary cause for therapy resistance in cancer as anti-cancer agents such as BRAF inhibitors also induce NRF2-dependent antioxidative response. We had reported that type I interferons produced upon activation of STING, abrogates NRF2 function. Therefore, we investigated if STING agonists such as the newly developed dimeric aminobenzimidazole (diABZI) could sensitize melanoma cells to the clinically used BRAF inhibitors. Our results reveal that pharmacological activation of STING by diABZI, down regulates NRF2-dependent anti-oxidative responses and potentiates cell-death in melanoma cells when used in combination with BRAF inhibitors.

Minutes of PPAR-γ agonism and neuroprotection.

Peroxisome proliferator-activated receptor gamma (PPAR-γ) is one of the ligand-activated transcription factors which regulates a number of central events and considered as a promising target for various neurodegenerative disease conditions. Numerous reports implicate that PPAR-γ agonists have shown neuroprotective effects by regulating genes transcription associated with the pathogenesis of neurodegeneration. In regards, this review critically appraises the recent knowledge of PPAR-γ receptors in neuroprotection in order to hypothesize potential neuroprotective mechanism of PPAR-γ agonism in chronic neurological conditions. Of note, the PPAR-γ's interaction dynamics with PPAR-γ coactivator-1α (PGC-1α) has gained significant attention for neuroprotection. Likewise, a plethora of studies suggest that the PPAR-γ pathway can be actuated by the endogenous ligands present in the CNS and thus identification and development of novel agonist for the PPAR-γ receptor holds a vow to prevent neurodegeneration. Together, the critical insights of this review enlighten the translational possibilities of developing novel neuroprotective therapeutics targeting PPAR-γ for various neurodegenerative disease conditions.