Regulation of mTORC1 by the Rag GTPases.

The Rag GTPases are an evolutionarily conserved family that play a crucial role in amino acid sensing by the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is often referred to as the master regulator of cell growth. mTORC1 hyperactivation is observed in multiple diseases such as cancer, obesity, metabolic disorders, and neurodegeneration. The Rag GTPases sense amino acid levels and form heterodimers, where RagA or RagB binds to RagC or RagD, to recruit mTORC1 to the lysosome where it becomes activated. Here, we review amino acid signaling to mTORC1 through the Rag GTPases.

SNAT7 regulates mTORC1 via macropinocytosis.

Mammalian target of rapamycin complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Some amino acids signal to mTORC1 through the Rag GTPase, whereas glutamine and asparagine activate mTORC1 through a Rag GTPase-independent pathway. Here, we show that the lysosomal glutamine and asparagine transporter SNAT7 activates mTORC1 after extracellular protein, such as albumin, is macropinocytosed. The N terminus of SNAT7 forms nutrient-sensitive interaction with mTORC1 and regulates mTORC1 activation independently of the Rag GTPases. Depletion of SNAT7 inhibits albumin-induced mTORC1 lysosomal localization and subsequent activation. Moreover, SNAT7 is essential to sustain KRAS-driven pancreatic cancer cell growth through mTORC1. Thus, SNAT7 links glutamine and asparagine signaling from extracellular protein to mTORC1 independently of the Rag GTPases and is required for macropinocytosis-mediated mTORC1 activation and pancreatic cancer cell growth.

G-Protein Coupled Receptor Signaling and Mammalian Target of Rapamycin Complex 1 Regulation.

The mammalian target of rapamycin (mTOR) senses upstream stimuli to regulate numerous cellular functions such as metabolism, growth, and autophagy. Increased activation of mTOR complex 1 (mTORC1) is typically observed in human disease and continues to be an important therapeutic target. Understanding the upstream regulators of mTORC1 will provide a crucial link in targeting hyperactivated mTORC1 in human disease. In this mini-review, we will discuss the regulation of mTORC1 by upstream stimuli, with a specific focus on G-protein coupled receptor signaling to mTORC1. SIGNIFICANCE STATEMENT: mTORC1 is a master regulator of many cellular processes and is often hyperactivated in human disease. Therefore, understanding the molecular underpinnings of G-protein coupled receptor signaling to mTORC1 will undoubtedly be beneficial for human disease.

Quantitative Longitudinal Imaging Reveals that Inhibiting Hedgehog Activity Alleviates the Hypoxic Tumor Landscape.

Metastases account for the majority of mortalities related to breast cancer. The onset and sustained presence of hypoxia strongly correlates with increased incidence of metastasis and unfavorable prognosis in patients with breast cancer. The Hedgehog (Hh) signaling pathway is dysregulated in breast cancer, and its abnormal activity enables tumor progression and metastasis. In addition to programming tumor cell behavior, Hh activity enables tumor cells to craft a metastasis-conducive microenvironment. Hypoxia is a prominent feature of growing tumors that impacts multiple signaling circuits that converge upon malignant progression. We investigated the role of Hh activity in crafting a hypoxic environment of breast cancer. We used radioactive tracer [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) to image tumor hypoxia. We show that tumors competent for Hh activity are able to establish a hypoxic milieu; pharmacologic inhibition of Hh signaling in a syngeneic mammary tumor model mitigates tumor hypoxia. Furthermore, in hypoxia, Hh activity is robustly activated in tumor cells and institutes increased HIF signaling in a VHL-dependent manner. The findings establish a novel perspective on Hh activity in crafting a hypoxic tumor landscape and molecularly navigating the tumor cells to adapt to hypoxic conditions. IMPLICATIONS: Importantly, we present a translational strategy of utilizing longitudinal hypoxia imaging to measure the efficacy of vismodegib in a preclinical model of triple-negative breast cancer.

AKAP13 couples GPCR signaling to mTORC1 inhibition.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.

Hedgehog Signaling Regulates Metabolism and Polarization of Mammary Tumor-Associated Macrophages.

Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited. SIGNIFICANCE: These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.

Normalizing glucose levels reconfigures the mammary tumor immune and metabolic microenvironment and decreases metastatic seeding.

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

An Emerging Regulatory Role for the Tumor Microenvironment in the DNA Damage Response to Double-Strand Breaks.

Radiation, alkylating agents, and platinum-based chemotherapy treatments eliminate cancer cells through the induction of excessive DNA damage. The resultant DNA damage challenges the cancer cell's DNA repair capacity. Among the different types of DNA damage induced in cells, double-strand breaks (DSB) are the most lethal if left unrepaired. Unrepaired DSBs in tumor cells exacerbate existing gene deletions, chromosome losses and rearrangements, and aberrant features that characteristically enable tumor progression, metastasis, and drug resistance. Tumor microenvironmental factors like hypoxia, inflammation, cellular metabolism, and the immune system profoundly influence DSB repair mechanisms. Here, we put into context the role of the microenvironment in governing DSB repair mechanisms.