IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling

• Text
• PDF

Abstract

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis–free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.

Authors

Jinchang Lu, Guohui Song, Qinglian Tang, Changye Zou, Feng Han, Zhiqiang Zhao, Bicheng Yong, Junqiang Yin, Huaiyuan Xu, Xianbiao Xie, Tiebang Kang, YingLee Lam, Huiling Yang, Jingnan Shen, Jin Wang

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

• Text
• PDF

Abstract

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers.

Authors

Jit Kong Cheong, Fuquan Zhang, Pei Jou Chua, Boon Huat Bay, Andrew Thorburn, David M. Virshup

Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment

• Text
• PDF

Abstract

The mechanistic target of rapamycin (mTOR) is hyperactivated in many types of cancer, rendering it a compelling drug target; however, the impact of mTOR inhibition on metabolic reprogramming in cancer is incompletely understood. Here, by integrating metabolic and functional studies in glioblastoma multiforme (GBM) cell lines, preclinical models, and clinical samples, we demonstrate that the compensatory upregulation of glutamine metabolism promotes resistance to mTOR kinase inhibitors. Metabolomic studies in GBM cells revealed that glutaminase (GLS) and glutamate levels are elevated following mTOR kinase inhibitor treatment. Moreover, these mTOR inhibitor–dependent metabolic alterations were confirmed in a GBM xenograft model. Expression of GLS following mTOR inhibitor treatment promoted GBM survival in an α-ketoglutarate–dependent (αKG-dependent) manner. Combined genetic and/or pharmacological inhibition of mTOR kinase and GLS resulted in massive synergistic tumor cell death and growth inhibition in tumor-bearing mice. These results highlight a critical role for compensatory glutamine metabolism in promoting mTOR inhibitor resistance and suggest that rational combination therapy has the potential to suppress resistance.

Authors

Kazuhiro Tanaka, Takashi Sasayama, Yasuhiro Irino, Kumi Takata, Hiroaki Nagashima, Naoko Satoh, Katsusuke Kyotani, Takashi Mizowaki, Taichiro Imahori, Yasuo Ejima, Kenta Masui, Beatrice Gini, Huijun Yang, Kohkichi Hosoda, Ryohei Sasaki, Paul S. Mischel, Eiji Kohmura

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

• Text
• PDF

Abstract

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β₃–binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

Authors

Wies van Roosmalen, Sylvia E. Le Dévédec, Ofra Golani, Marcel Smid, Irina Pulyakhina, Annemieke M. Timmermans, Maxime P. Look, Di Zi, Chantal Pont, Marjo de Graauw, Suha Naffar-Abu-Amara, Catherine Kirsanova, Gabriella Rustici, Peter A.C. ‘t Hoen, John W.M. Martens, John A. Foekens, Benjamin Geiger, Bob van de Water

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway

• Text
• PDF

Abstract

The Fanconi anemia/BRCA (FA/BRCA) pathway is a DNA repair pathway that is required for excision of DNA interstrand cross-links. The 17 known FA proteins, along with several FA-associated proteins (FAAPs), cooperate in this pathway to detect, unhook, and excise DNA cross-links and to subsequently repair the double-strand breaks generated in the process. In the current study, we identified a patient with FA with a point mutation in FANCA, which encodes a mutant FANCA protein (FANCA^I939S). FANCA^I939S failed to bind to the FAAP20 subunit of the FA core complex, leading to decreased stability. Loss of FAAP20 binding exposed a SUMOylation site on FANCA at amino acid residue K921, resulting in E2 SUMO-conjugating enzyme UBC9-mediated SUMOylation, RING finger protein 4–mediated (RNF4-mediated) polyubiquitination, and proteasome-mediated degradation of FANCA. Mutation of the SUMOylation site of FANCA rescued the expression of the mutant protein. Wild-type FANCA was also subject to SUMOylation, RNF4-mediated polyubiquitination, and degradation, suggesting that regulated release of FAAP20 from FANCA is a critical step in the normal FA pathway. Consistent with this model, cells lacking RNF4 exhibited interstrand cross-linker hypersensitivity, and the gene encoding RNF4 was epistatic with the other genes encoding members of the FA/BRCA pathway. Together, the results from our study underscore the importance of analyzing unique patient-derived mutations for dissecting complex DNA repair processes.

Authors

Jenny Xie, Hyungjin Kim, Lisa A. Moreau, Shannon Puhalla, Judy Garber, Muthana Al Abo, Shunichi Takeda, Alan D. D’Andrea

A versatile modular vector system for rapid combinatorial mammalian genetics

• Text
• PDF

Abstract

Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-Ras^G12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.

Authors

Joachim Albers, Claudia Danzer, Markus Rechsteiner, Holger Lehmann, Laura P. Brandt, Tomas Hejhal, Antonella Catalano, Philipp Busenhart, Ana Filipa Gonçalves, Simone Brandt, Peter K. Bode, Beata Bode-Lesniewska, Peter J. Wild, Ian J. Frew

Co-clinical assessment identifies patterns of BRAF inhibitor resistance in melanoma

• Text
• PDF

Abstract

Multiple mechanisms have been described that confer BRAF inhibitor resistance to melanomas, yet the basis of this resistance remains undefined in a sizable portion of patient samples. Here, we characterized samples from a set of patients with melanoma that included individuals at baseline diagnosis, on BRAF inhibitor treatment, and with resistant tumors at both the protein and RNA levels. Using RNA and DNA sequencing, we identified known resistance-conferring mutations in 50% (6 of 12) of the resistant samples. In parallel, targeted proteomic analysis by protein array categorized the resistant samples into 3 stable groups, 2 of which were characterized by reactivation of MAPK signaling to different levels and 1 that was MAPK independent. The molecular relevance of these classifications identified in patients was supported by both mutation data and the similarity of resistance patterns that emerged during a co-clinical trial in a genetically engineered mouse (GEM) model of melanoma that recapitulates the development of BRAF inhibitor resistance. Additionally, we defined candidate biomarkers in pre- and early-treatment patient samples that have potential for predicting clinical responses. On the basis of these observations, we suggest that BRAF inhibitor–resistant melanomas can be actionably classified using protein expression patterns, even without identification of the underlying genetic alteration.

Authors

Lawrence N. Kwong, Genevieve M. Boland, Dennie T. Frederick, Timothy L. Helms, Ahmad T. Akid, John P. Miller, Shan Jiang, Zachary A. Cooper, Xingzhi Song, Sahil Seth, Jennifer Kamara, Alexei Protopopov, Gordon B. Mills, Keith T. Flaherty, Jennifer A. Wargo, Lynda Chin

Coactivator SRC-2–dependent metabolic reprogramming mediates prostate cancer survival and metastasis

• Text
• PDF

Abstract

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2–driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer.

Authors

Subhamoy Dasgupta, Nagireddy Putluri, Weiwen Long, Bin Zhang, Jianghua Wang, Akash K. Kaushik, James M. Arnold, Salil K. Bhowmik, Erin Stashi, Christine A. Brennan, Kimal Rajapakshe, Cristian Coarfa, Nicholas Mitsiades, Michael M. Ittmann, Arul M. Chinnaiyan, Arun Sreekumar, Bert W. O’Malley

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

• Text
• PDF

Abstract

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21^CIP1/WAF1-deficient, K-ras^G12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

Authors

Yulong Chen, Masahiko Terajima, Yanan Yang, Li Sun, Young-Ho Ahn, Daniela Pankova, Daniel S. Puperi, Takeshi Watanabe, Min P. Kim, Shanda H. Blackmon, Jaime Rodriguez, Hui Liu, Carmen Behrens, Ignacio I. Wistuba, Rosalba Minelli, Kenneth L. Scott, Johannah Sanchez-Adams, Farshid Guilak, Debananda Pati, Nishan Thilaganathan, Alan R. Burns, Chad J. Creighton, Elisabeth D. Martinez, Tomasz Zal, K. Jane Grande-Allen, Mitsuo Yamauchi, Jonathan M. Kurie

Musashi2 sustains the mixed-lineage leukemia–driven stem cell regulatory program

• Text
• PDF

Abstract

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia.

Authors

Sun-Mi Park, Mithat Gönen, Ly Vu, Gerard Minuesa, Patrick Tivnan, Trevor S. Barlowe, James Taggart, Yuheng Lu, Raquel P. Deering, Nir Hacohen, Maria E. Figueroa, Elisabeth Paietta, Hugo F. Fernandez, Martin S. Tallman, Ari Melnick, Ross Levine, Christina Leslie, Christopher J. Lengner, Michael G. Kharas