HIF-1α, one of the most extensively studied oncogenes, is activated by a variety of microenvironmental factors. The resulting biological effects are thought to depend on its transcriptional activity. The RNAse enzyme Dicer is frequently downregulated in human cancers, which has been functionally linked to enhanced metastatic properties; however, current knowledge of the upstream mechanisms regulating Dicer is limited. In the present study, we identified Dicer as a HIF-1α–interacting protein in multiple types of cancer cell lines and different human tumors. HIF-1α downregulated Dicer expression by facilitating its ubiquitination by the E3 ligase Parkin, thereby enhancing autophagy-mediated degradation of Dicer, which further suppressed the maturation of known tumor suppressors, such as the microRNA let-7 and microRNA-200b. Consequently, expression of HIF-1α facilitated epithelial-mesenchymal transition (EMT) and metastasis in tumor-bearing mice. Thus, this study uncovered a connection between oncogenic HIF-1α and the tumor-suppressive Dicer. This function of HIF-1α is transcription independent and occurs through previously unrecognized protein interaction–mediated ubiquitination and autophagic proteolysis.
Hui-Huang Lai, Jie-Ning Li, Ming-Yang Wang, Hsin-Yi Huang, Carlo M. Croce, Hui-Lung Sun, Yu-Jhen Lyu, Jui-Wen Kang, Ching-Feng Chiu, Mien-Chie Hung, Hiroshi I. Suzuki, Pai-Sheng Chen
The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor–bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF–endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.
Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Samuel Y. Ho, Lingegowda S. Mangala, Anil K. Sood, Gabriel Lopez-Berestein, Jianting Sheng, Stephen T.C. Wong, Michael J. Birrer, Samuel C. Mok
DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle–regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1–stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.
Guo Chen, Andrew T. Magis, Ke Xu, Dongkyoo Park, David S. Yu, Taofeek K. Owonikoko, Gabriel L. Sica, Sarah W. Satola, Suresh S. Ramalingam, Walter J. Curran, Paul W. Doetsch, Xingming Deng
SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.
Hironari Tamiya, Hyungsoo Kim, Oleksiy Klymenko, Heejung Kim, Yongmei Feng, Tongwu Zhang, Ji Yun Han, Ayako Murao, Scott J. Snipas, Lucia Jilaveanu, Kevin Brown, Harriet Kluger, Hao Zhang, Kazuhiro Iwai, Ze’ev A. Ronai
γδT cells produce inflammatory cytokines and have been implicated in the pathogenesis of cancer, infectious diseases, and autoimmunity. The T cell receptor (TCR) signal transduction that specifically regulates the development of IL-17–producing γδT (γδT17) cells largely remains unclear. Here, we showed that the receptor proximal tyrosine kinase Syk is essential for γδTCR signal transduction and development of γδT17 in the mouse thymus. Zap70, another tyrosine kinase essential for the development of αβT cells, failed to functionally substitute for Syk in the development of γδT17. Syk induced the activation of the PI3K/Akt pathway upon γδTCR stimulation. Mice deficient in PI3K signaling exhibited a complete loss of γδT17, without impaired development of IFN-γ–producing γδT cells. Moreover, γδT17-dependent skin inflammation was ameliorated in mice deficient in RhoH, an adaptor known to recruit Syk. Thus, we deciphered lineage-specific TCR signaling and identified the Syk/PI3K pathway as a critical determinant of proinflammatory γδT cell differentiation.
Ryunosuke Muro, Takeshi Nitta, Kenta Nakano, Tadashi Okamura, Hiroshi Takayanagi, Harumi Suzuki
The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.
Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen
The incorporation of excess saturated free fatty acids (SFAs) into membrane phospholipids within the ER promotes ER stress, insulin resistance, and hepatic gluconeogenesis. Thioesterase superfamily member 2 (Them2) is a mitochondria-associated long-chain fatty acyl-CoA thioesterase that is activated upon binding phosphatidylcholine transfer protein (PC-TP). Under fasting conditions, the Them2/PC-TP complex directs saturated fatty acyl-CoA toward β-oxidation. Here, we showed that during either chronic overnutrition or acute induction of ER stress, Them2 and PC-TP play critical roles in trafficking SFAs into the glycerolipid biosynthetic pathway to form saturated phospholipids, which ultimately reduce ER membrane fluidity. The Them2/PC-TP complex activated ER stress pathways by enhancing translocon-mediated efflux of ER calcium. The increased cytosolic calcium, in turn, led to the phosphorylation of calcium/calmodulin-dependent protein kinase II, which promoted both hepatic insulin resistance and gluconeogenesis. These findings delineate a mechanistic link between obesity and insulin resistance and establish the Them2/PC-TP complex as an attractive target for the management of hepatic steatosis and insulin resistance.
Baran A. Ersoy, Kristal M. Maner-Smith, Yingxia Li, Ipek Alpertunga, David E. Cohen
p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an “obligatory” haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3–dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.
Sarah P. Short, Jumpei Kondo, Whitney G. Smalley-Freed, Haruna Takeda, Michael R. Dohn, Anne E. Powell, Robert H. Carnahan, Mary K. Washington, Manish Tripathi, D. Michael Payne, Nancy A. Jenkins, Neal G. Copeland, Robert J. Coffey, Albert B. Reynolds
Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M– and IL-6–triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.
Shouyu Wang, Ke Liang, Qingsong Hu, Ping Li, Jian Song, Yuedong Yang, Jun Yao, Lingegowda Selanere Mangala, Chunlai Li, Wenhao Yang, Peter K. Park, David H. Hawke, Jianwei Zhou, Yan Zhou, Weiya Xia, Mien-Chie Hung, Jeffrey R. Marks, Gary E. Gallick, Gabriel Lopez-Berestein, Elsa R. Flores, Anil K. Sood, Suyun Huang, Dihua Yu, Liuqing Yang, Chunru Lin
Astrocytes perform critical non–cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.
Izhar Livne-Bar, Jessica Wei, Hsin-Hua Liu, Samih Alqawlaq, Gah-Jone Won, Alessandra Tuccitto, Karsten Gronert, John G. Flanagan, Jeremy M. Sivak
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