Autocrine IGF1 Signaling Mediates Pancreatic Tumor Cell Dormancy in the Absence of Oncogenic Drivers.
ABSTRACT: Mutant KRAS and c-MYC are oncogenic drivers and rational therapeutic targets for the treatment of pancreatic cancer. Although tumor growth and homeostasis are largely dependent on these oncogenes, a few residual cancer cells are able to survive the ablation of mutant KRAS and c-MYC. By performing a genome-wide gene expression analysis of in vivo-derived bulk tumor cells and residual cancer cells lacking the expression of mutant KRAS or c-MYC, we have identified an increase in autocrine IGF1/AKT signaling as a common survival mechanism in dormant cancer cells. The pharmacological inhibition of IGF-1R reduces residual disease burden and cancer recurrence, suggesting that this molecular pathway is crucial for the survival of cancer cells in the absence of the primary oncogenic drivers.
Project description:Mutant KRAS and c-MYC are oncogenic drivers and rational therapeutic targets for the treatment of pancreatic cancer. While tumor growth and homeostasis is largely dependent on these oncogenes, a few residual cancer cells are able to survive the ablation of mutant KRAS and c-MYC. By performing a genome-wide gene expression analysis of in vivo-derived bulk tumor cells and residual cancer cells lacking the expression of mutant KRAS or c-MYC, we have identified an increase in autocrine IGF1/AKT signaling as a common survival mechanism in dormant cancer cells. The pharmacological inhibition of IGF-1R reduced residual disease burden and cancer recurrence, suggesting this molecular pathway is crucial for the survival of cancer cells in the absence of the primary oncogenic drivers. Overall design: Total RNA was extracted from flash-frozen pancreatic cancer tissues before or after doxycycline treatment of tumor transplanted athymic nude mice using the RNeasy Mini Kit (Qiagen).
Project description:Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway.
Project description:KRAS is the most commonly mutated oncogene, frequently associated with some of the deadliest forms of cancer. However, the need for potent and specific KRAS inhibitors remains unmet. Here, we evaluated the effects of selected cytotoxic agents on oncogenic KRAS signaling and drug response. The data provided new insights into the functional interaction between the KRAS and MYC pathways and revealed key differences between WT and mutant KRAS expressing cells. Systematic investigation of non-small cell lung cancer cell lines revealed that KRAS mutation can paradoxically increase the sensitivity of cells to cytotoxic agents. We identify MYC as a key regulator of the cellular stress responses and tumor cell viability as MYC expression was suppressed in drug-sensitive but not resistant cells. Furthermore, this suppression was driven by hyperactive KRAS/MAPK signaling. Our findings support a direct link between MYC and cancer cell viability, and raise the possibility that inactivation of MYC may be an effective therapeutic strategy for KRAS mutant tumors across various cancer types.
Project description:Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs.
Project description:Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.
Project description:Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-?B transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently, TGF-?-activated kinase 1 (TAK1), an upstream regulator of I?B kinase (IKK), which controls canonical NF-?B signaling, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS-mutant colorectal cancer cell growth and survival. Here, we show that mutant KRAS upregulates glycogen synthase kinase 3? (GSK-3?), leading to its interaction with TAK1 to stabilize the TAK1-TAB complex to promote IKK activity. In addition, GSK-3? is required for promoting critical noncanonical NF-?B signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3? as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-?B signaling pathways.
Project description:Non-small cell lung cancer (NSCLC) is often characterized by mutually exclusive mutations in the epidermal growth factor receptor (EGFR) or the guanosine triphosphatase KRAS. We hypothesized that blocking EGFR palmitoylation, previously shown to inhibit EGFR activity, might alter downstream signaling in the KRAS-mutant setting. Here, we found that blocking EGFR palmitoylation, by either knocking down the palmitoyltransferase DHHC20 or expressing a palmitoylation-resistant EGFR mutant, reduced activation of the kinase PI3K, the abundance of the transcription factor MYC, and the proliferation of cells in culture, as well as reduced tumor growth in a mouse model of KRAS-mutant lung adenocarcinoma. Knocking down DHHC20 reduced the growth of existing tumors derived from human KRAS-mutant lung cancer cells and increased the sensitivity of these cells to a PI3K inhibitor. Palmitoylated EGFR interacted with the PI3K regulatory subunit PIK3R1 (p85) and increased the recruitment of the PI3K heterodimer to the plasma membrane. Alternatively, blocking palmitoylation increased the association of EGFR with the MAPK adaptor Grb2 and decreased that with p85. This binary switching between MAPK and PI3K signaling, modulated by EGFR palmitoylation, was only observed in the presence of oncogenic KRAS. These findings suggest a mechanism whereby oncogenic KRAS saturates signaling through unpalmitoylated EGFR, reducing formation of the PI3K signaling complex. Future development of DHHC20 inhibitors to reduce EGFR-PI3K signaling could be beneficial to patients with KRAS-mutant tumors.
Project description:Ammonium tetrathiomolybdate (TTM) and disulfiram (DSF) are copper (Cu) chelators in cancer clinical trials partly because Cu chelation: a) restricts the activity of Cu-binding MEK1/2 enzymes which drive tumourigenesis by KRAS or BRAF oncogenic mutations and b) enhances uptake of oxaliplatin (OxPt), clinically used in advanced KRAS-mutant colorectal carcinomas (CRC). Whereas TTM decreases intracellular Cu trafficking, DSF can reach other Cu-dependent intracellular proteins. Since the use of individual or combined Cu chelation may help or interfere with anti-cancer therapy, this study investigated whether TTM modifies the response to DSF supplemented with: 1) UO126, a known MEK1/2 inhibitor; 2) other Cu chelators like neocuproine (NC) or 1, 10-o-phenanthroline (OPT) in wt p53 melanoma cells differing in BRAF or KRAS mutations; 3) OxPt in mutant p53 CRC cells devoid of KRAS and BRAF mutations or harbouring either KRAS or BRAF mutations. TTM was not toxic against V600E- mut-BRAF A375 and G12D-mut- KRAS/high c-myc C8161 melanoma cells. Moreover, TTM protected both melanoma types from toxicity induced by DSF, NC and co-treatment with sub-lethal levels of DSF and the MEK inhibitor, UO126. Toxicity by co-treatment with DSF+OPT was poorly reversed by TTM in C8161 melanoma cells. In contrast to the greater toxicity of 0.1 ?M DSF against mutant p53 CRC cells irrespective of their KRAS mutation, TTM did not protect G12V-mut- KRAS/high c-myc SW620 CRC from DSF+OxPt compared to KRAS-WT/BRAF-WT Caco-2 CRC. Our results show that DSF co-treatment with: a) MEK inhibitors may enhance tumour suppression; b) OxPt in CRC may counteract impaired response to cetuximab by KRAS/BRAF mutations and c) as a single treatment, TTM may be less effective than DSF and decreases the efficacy of the latter. Highlights:Potentiation of melanoma antitumour toxicity of DSF by MEK inhibitor is reversed by TTM.KRAS/c-MYC dysregulation attenuates TTM reversion of melanoma toxicity by DSF + OPT.KRAS/c-MYC dysregulation increases melanoma NC toxicity reversed by TTM.BRAF mutation and lower c-MYC may attenuate toxicity by DSF ± OxPt in colorectal cancer cells.
Project description:Aberrant regulation of the Wnt/?-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/?-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in ?-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (?NTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced ?-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished ?-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting ?-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/?-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/?-catenin signalings during oncogenesis.
Project description:Dysregulation of MYC expression is a hallmark of cancer, but the development of agents that target MYC has remained challenging. The oncogenic MUC1-C transmembrane protein is, like MYC, aberrantly expressed in diverse human cancers. The present studies demonstrate that MUC1-C induces MYC expression in KRAS mutant non-small cell lung cancer (NSCLC) cells, an effect that can be suppressed by targeting MUC1-C via shRNA silencing, CRISPR editing, or pharmacologic inhibition with GO-203. MUC1-C activated the WNT/?-catenin (CTNNB1) pathway and promoted occupancy of MUC1-C/?-catenin/TCF4 complexes on the MYC promoter. MUC1-C also promoted the recruitment of the p300 histone acetylase (EP300) and, in turn, induced histone H3 acetylation and activation of MYC gene transcription. We also show that targeting MUC1-C decreased the expression of key MYC target genes essential for the growth and survival of NSCLC cells, such as TERT and CDK4. Based on these results, we found that the combination of GO-203 and the BET bromodomain inhibitor JQ1, which targets MYC transcription, synergistically suppressed MYC expression and cell survival in vitro as well as tumor xenograft growth. Furthermore, MUC1 expression significantly correlated with that of MYC and its target genes in human KRAS mutant NSCLC tumors. Taken together, these findings suggest a therapeutic approach for targeting MYC-dependent cancers and provide the framework for the ongoing clinical studies addressing the efficacy of MUC1-C inhibition in solid tumors.