De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.
ABSTRACT: Oncogenic Kras mutations are one of the most common alterations in non-small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up-regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras-mutant lung cancer. Using the Kras LSL-G12D lung cancer model, we show that mutant Kras drives a lipogenic gene-expression program. Stable-isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras-induced tumorigenesis was evident by decreased tumor formation in Kras LSL-G12D mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.-Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah-Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.-k., Haley, J. D., Biswal, S., Girnun, G. D. De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.
Project description:We examine the potential of Kras as a metabolic target in lung cancer using the KrasLSL-G12D lung cancer model. We demonstrate that mutant Kras drives a lipogenic gene expression program, and that fatty acid synthesis is important in Kras-induced tumorigenesis. Compare gene expression changes between mutant Kras (G12D) driven lung tumors and normal lung samples to identify pathways selectively altered in lung tumors. Overall design: To identify the pathways selectively modulated by oncogenic Kras (G12D) signaling in lung tumors, we used the transgenic Kras LSL-G12D model of lung cancer. By crossing these mice with another transgenic mice (CC10_Cre) expressing Cre recombinase selectively in Clara cells of the lung, we generated Kras LSL-G12D CC10-Cre mice which selectively express mutant kras in clara cells. These mice develop lung adenocarcinoma and have a median survival of 80 days.
Project description:Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1) transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC). Six-week-old female p48(Cre/+)-LSL-Kras(G12D/+) and compound Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice were fed (AIN-76A) diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P < .02) in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. Importantly, significant reductions of pancreatic ducts with carcinoma (90%; P < .0001) and PanIN 3 (~50%; P < .001) lesions were observed in the compound transgenic mice. The levels of n-3 PUFA were much higher (>85%; P < .05-0.01) in pancreas of compound transgenic mice than in those of p48(Cre/+)-LSL-Kras(G12D/+) mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX), 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.
Project description:Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRAS<sup>G12D</sup> mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (Kras<sup>LSL-G12D</sup>) alleles. Simultaneous activation of the Kras<sup>G12D</sup> allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress Kras<sup>G12D</sup>-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (Kras<sup>G12D/+</sup>) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.
Project description:Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-?B transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the I?B? inhibitor or by genetic deletion of IKK? or the RELA/p65 subunit of NF-?B. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKK? inhibitor Compound A (CmpdA) led to NF-?B inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKK? or IKK? reduced activity of the NF-?B canonical pathway. Next, we determined that both IKK? and IKK? contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-Kras (G12D)) combined with loss of p53 (LSL-Kras (G12D)/p53 (fl/fl)). CmpdA treatment was well tolerated and mice treated with this IKK? inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKK? inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity.
Project description:Lung cancer is the leading cause of cancer deaths worldwide, and current therapies fail to treat this disease in the vast majority of cases. The RAS and p53 pathways are two of the most frequently altered pathways in lung cancers, with such alterations resulting in loss of responsiveness to current therapies and decreased patient survival. The microRNA-34 (mir-34) gene family members are downstream transcriptional targets of p53, and miR-34 expression is reduced in p53 mutant tumors; thus, we hypothesized that treating mutant Kras;p53 tumors with miR-34 would represent a powerful new therapeutic to suppress lung tumorigenesis. To this end we examined the therapeutically resistant Kras(LSL-G12D)(/+);Trp53(LSL-R172H)(/+) mouse lung cancer model. We characterized tumor progression in these mice following lung-specific transgene activation and found tumors as early as 10 weeks postactivation, and severe lung inflammation by 22 weeks. Tumors harvested from these lungs have elevated levels of oncogenic miRNAs, miR-21 and miR-155; are deficient for p53-regulated miRNAs; and have heightened expression of miR-34 target genes, such as Met and Bcl-2. In the presence of exogenous miR-34, epithelial cells derived from these tumors show reduced proliferation and invasion. In vivo treatment with miR-34a prevented tumor formation and progression in Kras(LSL-G12D)(/+);Trp53(LSL-R172H)(/+) mice. Animals infected with mir-34a-expressing lentivirus at the same time as transgene activation had little to no evidence of tumorigenesis, and lentivirus-induced miR-34a also prevented further progression of preformed tumors. These data support the use of miR-34 as a lung tumor-preventative and tumor-static agent.
Project description:Activin, a member of the transforming growth factor-? (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis.We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry.Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades.Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer.
Project description:NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.
Project description:<h4>Background</h4>Methionyl-tRNA synthetase (MRS) plays a critical role in initiating translation by transferring Met to the initiator tRNA (tRNA<sub>i</sub><sup>Met</sup>) and protection against ROS-mediated damage, suggesting that its overexpression is related to cancer growth and drug resistance. In this study, the clinical implication of MRS expression in non-small cell lung cancer (NSCLC) was evaluated.<h4>Methods</h4>Immunoblot and immunohistochemical (IHC) analyses were performed using tissue lysates and formalin-fixed paraffin embedded (FFPE) tissue blocks from wild type C57BL/6, LSL-Kras G12D, and LSL-Kras G12D:p53<sup>fl/fl</sup> mice. For human studies, 12 paired adjacent normal appearing lung tissue lysates and cancer tissue lysates, in addition to 231 FFPE tissue samples, were used.<h4>Results</h4>MRS was weakly expressed in the spleen and intestinal epithelium and only marginally expressed in the kidney, liver, and lungs of wild type C57BL/6 mice. On the other hand, MRS was strongly expressed in the neoplastic region of lung tissue from LSL-Kras G12D and LSL-Kras G12D:p53<sup>fl/fl</sup> mice. Immunoblot analysis of the human normal appearing adjacent and lung cancer paired tissue lysates revealed cancer-specific MRS overexpression, which was related to mTORC1 activity. IHC analysis of the 231 FFPE lung cancer tissue samples showed that MRS expression was frequently detected in the cytoplasm of lung cancer cells (179 out of 231, 77.4%), with a small proportion (73 out of 231, 31.6%) also showing nuclear expression. The proportion of cases with positive MRS expression was higher in the advanced pStage subgroup (P = 0.018, ?<sup>2</sup>-test) and cases with MRS expression also had shorter DFS (161.6 vs 142.3, P = 0.014, log-rank test).<h4>Conclusions</h4>Taken together, MRS is frequently overexpressed in NSCLC. Moreover, MRS is related to mTORC1 activity and its overexpression is associated with poor clinical outcomes, indicating that it has potential as a putative therapeutic target.