Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2−/− (tuberous sclerosis complex 2−/−) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an ATP-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24- CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.

Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression.

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression, and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. Since miR-124 expression is repressed in various cancers (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival, during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared to regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo, and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 re-introduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

Key Points• ARNT promotes adult hematopoietic stem cell viability through regulation of BCL-2 and VEGF-A expression.• Fetal liver hematopoietic progenitors experience hypoxia and loss of hypoxiainduced transcription decreases their survival.Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1a promotes HSC adaptation to stress. Because the related factor HIF-2a is also expressed in HSCs, the combined role of HIF-1a and HIF-2a in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-a dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1a and HIF-2a and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1a and Hif-2a phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors. (Blood. 2015;125(21):3263-3272) IntroductionHematopoietic stem cells (HSCs) reside in the bone marrow (BM), where they balance both cell-intrinsic and cell-extrinsic cues to achieve self-renewal and appropriate hematologic differentiation throughout the mammalian lifespan.1 HSCs are regulated by their microenvironment, which consists of endothelial, 2 perivascular, 2 adipocyte, 3 and osteoblast 4 support cells; secreted factors; and oxygen (O 2 ) availability. 5Hypoxia has become increasingly recognized as a critical regulator of stem cells, during both embryonic development and adulthood. 6 Importantly, HSCs reside in a poorly perfused hypoxic niche, [7][8][9][10] and recent data suggest that HSC oxygenation levels may be partially regulated by cell-specific mechanisms. 10 Although the biological importance of these observations is not entirely clear, hypoxia clearly imposes phenotypic consequences for HSCs. For instance, long-term HSCs (LT-HSCs) are highly quiescent, a cell cycle status often associated with O 2 -and nutrient-deprived cells. Although many pathways converge on metabolism, the primary transcriptional response to hypoxia is mediated by hypoxia-inducible factors (HIFs).HIFs are heterodimeric transcription factors composed of a HIF-a subunit ) and their common b subunit, HIF-1b or the aryl hydrocarbon receptor nuclear translocator (ARNT).13 HIFa/ARNT heterodimers stimulate the transcription of many genes, which promote survival and adaptation to ...

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mTORC1 activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2-/- cells deprived of serum lipids exhibit a magnified UPR response, but fail to appropriately expand their ER, leading to IRE1-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observe this phenotype in multiple human cancer lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids. Citation Format: Regina Young, Daniel Ackerman, Zachary Quinn, Anthony Mancuso, Michaela Gruber, Liping Liu, Dionysios Giannoukos, Ekaterina Bobovnikova-Marjon, J. Alan Diehl, Brian Keith, M. Celeste Simon. Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA16.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis and lacks targeted therapies. Basal-like TNBC exhibits hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation of different cancers. However, it is unknown whether JNK signaling is a clinically relevant target in TNBC. Here, we tested the hypotheses that JNK signaling plays a fundamental role in TNBC pathogenesis by promoting self-renewal of cancer stem cells (CSCs). Methods: The role of JNK signaling in TNBC pathogenesis was determined by examining the effects of JNK signaling inhibition using siRNA or JNK-IN-8 (an ATP-competitive JNK inhibitor) and the effects of JNK overexpression on growth, migration, and invasion of TNBC cells using cell proliferation, migration, and invasion assays. CSC regulation by JNK signaling was examined using mammosphere-forming assay. The effect of JNK signaling inhibition on tumor growth was assessed using a xenograft mouse model. The downstream molecules involved in JNK signaling-mediated CSC regulation were identified using RT-PCR stem array analysis. Association between JNK and c-Jun was analyzed using a reverse phase protein array dataset of TNBC samples (n = 81). Disease-free survival (DFS) probability in TNBC patients was analyzed by the Kaplan-Meier method using a cDNA microarray dataset (n = 79). Results: Knockdown of JNK1 or JNK2 led to significant reductions in growth, migration, and invasion of TNBC cells, mammosphere formation, and ALDH1+ CSC subpopulation. Similar results were obtained when JNK signaling was inhibited using JNK-IN-8. In contrast, overexpression of JNK1 or JNK2 enhanced these cellular activities. Importantly, JNK signaling inhibition by JNK-IN-8 (25 mg/kg) reduced tumor growth by 60.88% (P<0.001) in a xenograft mouse model. RT-PCR stem array analysis indicated the involvement of Notch1 in JNK signaling-mediated CSC regulation; luciferase reporter assay showed that JNK regulated Notch1 expression at the transcriptional level via c-Jun, a transcription factor and a substrate of JNK. Knockdown of c-Jun or Notch1 reduced cell growth, mammosphere formation, and ALDH1+ CSC subpopulation; overexpression of c-Jun or Notch1 blocked the inhibitory effect of JNK-IN-8 on mammosphere formation. These results suggest that JNK signaling promotes CSC self-renewal through up-regulation of Notch1 via c-Jun. Furthermore, in TNBC patients, a high level of phosphorylated JNK was associated with a high level of phosphorylated c-Jun (P = 3×10-8); low c-Jun mRNA level was associated with better DFS (P = 0.03); and there was a tendency (P = 0.17) toward better DFS with low JNK1 mRNA level but not with low JNK2 mRNA level. Conclusion: JNK signaling regulates TNBC tumorigenesis by promoting CSC self-renewal via Notch1 signaling. JNK-IN-8 is a promising therapeutic agent for TNBC by targeting JNK/Notch1 signaling. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Dionysios N. Giannoukos, Yuan Qi, Debu Tripathy, Nathanael S. Gray, Kevin N. Dalby, Chandra Bartholomeusz, Naoto T. Ueno. Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2015-1505