Glutamine is essential for overcoming the immunosuppressive microenvironment in malignant salivary gland tumors.

Rationale: Immunosuppression in the tumor microenvironment (TME) is key to the pathogenesis of solid tumors. Tumor cell-intrinsic autophagy is critical for sustaining both tumor cell metabolism and survival. However, the role of autophagy in the host immune system that allows cancer cells to escape immune destruction remains poorly understood. Here, we determined if attenuated host autophagy is sufficient to induce tumor rejection through reinforced adaptive immunity. Furthermore, we determined whether dietary glutamine supplementation, mimicking attenuated host autophagy, is capable of promoting antitumor immunity. Methods: A syngeneic orthotopic tumor model in Atg5+/+ and Atg5flox/flox mice was established to determine the impact of host autophagy on the antitumor effects against mouse malignant salivary gland tumors (MSTs). Multiple cohorts of immunocompetent mice were used for oncoimmunology studies, including inflammatory cytokine levels, macrophage, CD4+, and CD8+ cells tumor infiltration at 14 days and 28 days after MST inoculation. In vitro differentiation and in vivo dietary glutamine supplementation were used to assess the effects of glutamine on Treg differentiation and tumor expansion. Results: We showed that mice deficient in the essential autophagy gene, Atg5, rejected orthotopic allografts of isogenic MST cells. An enhanced antitumor immune response evidenced by reduction of both M1 and M2 macrophages, increased infiltration of CD8+ T cells, elevated IFN-γ production, as well as decreased inhibitory Tregs within TME and spleens of tumor-bearing Atg5flox/flox mice. Mechanistically, ATG5 deficiency increased glutamine level in tumors. We further demonstrated that dietary glutamine supplementation partially increased glutamine levels and restored potent antitumor responses in Atg5+/+ mice. Conclusions: Dietary glutamine supplementation exposes a previously undefined difference in plasticity between cancer cells, cytotoxic CD8+ T cells and Tregs.

Arginine starvation kills tumor cells through aspartate exhaustion and mitochondrial dysfunction.

Defective arginine synthesis, due to the silencing of argininosuccinate synthase 1 (ASS1), is a common metabolic vulnerability in cancer, known as arginine auxotrophy. Understanding how arginine depletion kills arginine-auxotrophic cancer cells will facilitate the development of anti-cancer therapeutic strategies. Here we show that depletion of extracellular arginine in arginine-auxotrophic cancer cells causes mitochondrial distress and transcriptional reprogramming. Mechanistically, arginine starvation induces asparagine synthetase (ASNS), depleting these cancer cells of aspartate, and disrupting their malate-aspartate shuttle. Supplementation of aspartate, depletion of mitochondria, and knockdown of ASNS all protect the arginine-starved cells, establishing the causal effects of aspartate depletion and mitochondrial dysfunction on the arginine starvation-induced cell death. Furthermore, dietary arginine restriction reduced tumor growth in a xenograft model of ASS1-deficient breast cancer. Our data challenge the view that ASNS promotes homeostasis, arguing instead that ASNS-induced aspartate depletion promotes cytotoxicity, which can be exploited for anti-cancer therapies.

Current State of Knowledge on Salivary Gland Cancers.

Salivary gland cancers (SGCs), categorized as head and neck cancers (HNCs), constitute about 6% of head and neck cancer diagnoses based on estimate by American Head and Neck Society. Salivary gland tumors originate from different glandular cell types and are thus morphologically diverse. These tumors arise from any of the three major and various minor salivary glands. The incidence of SGCs has slowly increased during the last four decades. The etiology of SGCs is mostly unknown; however, specific gene mutations are associated with certain types of salivary tumors. Treatment options include surgical resection, radiation therapy (RT), chemotherapy, and multimodality therapy. HNC patients treated with RT often develop xerostomia and salivary hypofunction due to damaged salivary glands. In this review, we discuss etiology of SGCs, present findings on the role of autophagy in salivary tumorigenesis, review adverse effects of radiation treatment, and examine remedies for restoration of salivary function.

Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis.

Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene Atg5 (atg5-KO) extends the survival of KRASG12V-driven tumor-bearing mice by 38%. atg5-KO tumors spread more slowly during late tumorigenesis, despite a faster onset. atg5-KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for de novo asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRASG12V-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.

An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid ß-Oxidation in Breast Cancer Cells.

The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid ß-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid ß-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction.

Identification of an AAA ATPase VPS4B-dependent pathway that modulates epidermal growth factor receptor abundance and signaling during hypoxia.

VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4B (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic-hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer.

SUMOylation of the transcriptional co-repressor KAP1 is regulated by the serine and threonine phosphatase PP1.

Krüppel-associated box (KRAB) domain-associated protein 1 [KAP1, also known as transcription intermediary factor-1beta (TIF1beta)] is a ubiquitous transcriptional co-repressor that is susceptible to phosphorylation at Ser(824) by ataxia-telangiectasia mutated (ATM) and to modification by small ubiquitin-like modifying (SUMO) proteins. Here, we found that, whereas the protein phosphatase 1alpha isoform (PP1alpha) directly interacted with KAP1 under basal conditions, PP1beta interacted with KAP1 only in response to genotoxic stress. Changes in the abundance of PP1alpha or PP1beta had differential effects on the phosphorylation and SUMOylation states of KAP1 under basal conditions and in response to DNA double-strand breaks (DSBs). Chromatin immunoprecipitation and re-immunoprecipitation experiments revealed that PP1alpha and PP1beta were recruited to KAP1 with different kinetics before and after the induction of DNA DSBs, which provided a mechanistic basis for the switch in the phosphorylation and SUMOylation states of KAP1. PP1beta-dependent SUMOylation of KAP1 occurred by mechanisms that were dependent and independent of the phosphorylation status of Ser(824). We posit a mechanism whereby the combined actions of PP1alpha and PP1beta cause dephosphorylation of KAP1 at Ser(824) and assure its SUMOylation to counter the effect of ATM, thereby regulating the transcription of KAP1 target genes in unstressed and stressed cells.

Suppression of nonhomologous end joining repair by overexpression of HMGA2.

Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and effect on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the nonhomologous end joining (NHEJ) process. We showed that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80, and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSB) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (gamma-H2AX) was associated with hyperphosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis.

SUMOylation of HMGA2: selective destabilization of promyelocytic leukemia protein via proteasome.

The HMGA2 architectural protein functions in a variety of cellular processes, such as cell growth, transcription regulation, neoplastic transformation, and progression. Up-regulation of HMGA2 protein is observed in many tumors and is associated with advanced cancers with poor prognoses. Although the expression and biochemical properties of HMGA2 protein are regulated by microRNA and phosphorylation, it is unknown whether HMGA2 activity can also be regulated by SUMOylation, and that is what is investigated in this report. We identified HMGA2 as a SUMOylation target and showed that the expression of wild-type HMGA2, but not SUMOylation-defective HMGA2(2K/R), selectively lowered the steady-state level of PML protein. Consequently, the HMGA2-elicited PML down-regulation rendered a reduction in the average number of PML nuclear bodies per cell and the volume of PML assembled per PML nuclear body. Using small interfering RNA to suppress endogenous ubiquitin expression and proteasome inhibitor to repress ubiquitin-mediated protein degradation, we showed that HMGA2 confers PML down-regulation through ubiquitin-proteasome-dependent protein degradation. Importantly, arsenic trioxide treatment stimulated HMGA2 SUMOylation, leading to the formation of HMGA2 nuclear foci surrounding PML nuclear bodies and the stimulation of PML degradation. Collectively, our results unveil a previously unrecognized effect by HMGA2 on the modulation of PML protein level, providing a novel mechanism underlying HMGA2 function and underscoring the molecular basis for oncogenic progression by HMGA2.

Transcriptional regulation by targeted expression of architectural transcription factor high mobility group A2 in salivary glands of transgenic mice.

High mobility group A2 (HMGA2) protein is a non-histone architectural transcription factor. Numerous studies have demonstrated that HMGA2 is exclusively expressed in the nucleus of embryonic, but not of terminally differentiated, cells, and aberrant expression of HMGA2 is associated with various benign tumors, including pleomorphic salivary adenoma. Herein, we report the use of a 4.5-kb enhancer/promoter region of the aquaporin-5 (AQP-5) gene to target HMGA2 transgene expression in the mouse salivary acinar cells as a model to investigate the biochemical and biological role of ectopic HMGA2 expression. The expression pattern was analyzed by microarray analyses to profile HMGA2-dependent salivary gene regulation. By using quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, the expression of a cluster of genes involved in cytokine signaling, including Il7r, Il2rg, and Ptprc, was verified to be up-regulated in the salivary glands of AQP-5/HMGA2 mice. In concert, the expression of a cluster of genes, namely Ppara, Phyh, and Cidea, governing fatty acid and lipid metabolism, was confirmed to be down-regulated by HMGA2. Additionally, squamous carcinoma-like salivary tumors were observed in the AQP-5/HMGA2 transgenic mice, albeit at a low incidence. Our findings indicate that the AQP-5 promoter/enhancer-containing region is sufficient to target salivary-specific transgene expression and suggest novel roles for HMGA2 in salivary epithelial cells.

High mobility group A2 potentiates genotoxic stress in part through the modulation of basal and DNA damage-dependent phosphatidylinositol 3-kinase-related protein kinase activation.

The high mobility group A2 (HMGA2) protein belongs to the architectural transcription factor HMGA family, playing a role in chromosomal organization and transcriptional regulation. We and others have previously reported that ectopic HMGA2 expression is associated with neoplastic transformation and anchorage-independent cell proliferation. Here, we reported a correlation between increased HMGA2 expression and enhanced chemosensitivity towards topoisomerase II inhibitor, doxorubicin, in breast cancer cells. Using cells exhibiting differential HMGA2 expression and small interfering RNA technique, we showed that HMGA2 expression modulates cellular response to the genotoxicity of DNA double-strand breaks. Notably, HMGA2 enhances doxorubicin-elicited cell cycle delay in sub-G1 and G2-M and augments cell cycle dysregulation on cotreatment of doxorubicin and caffeine. We further reported that HMGA2 induces a persistent Ser139 phosphorylation of histone 2A variant X, analogous to the activation by doxorubicin-mediated genotoxic stress. Moreover, this HMGA2-dependent enhancement of cytotoxicity is further extended to other double-strand breaks elicited by cisplatin and X-ray irradiation and is not restricted to one cell type. Together, we postulated that the enhanced cytotoxicity by double-strand breaks in HMGA2-expressing cells is mediated, at least in part, through the signaling pathway of which the physiologic function is to maintain genome integrity. These findings should contribute to a greater understanding of the role of HMGA2 in promoting tumorigenesis and conveying (chemo)sensitivity towards doxorubicin and other related double-strand breaks.