PDF file - 161K, Ctrl and MLK4 knock down DLD were seeded and allowed to grow for the indicated times in flasks with ultra-low attachment surface to prevent cell adhesion. Cells were harvested and protein lysates were subjected to western blotting with the indicated antibodies.

PDF file - 422K, A) Ctrl and MLK4 knock down (Sh1 and Sh2) Sw48 cells were stimulated with EGF (2nM-20 nM) at 37 {degree sign}C for 10 minutes. Cell lysates were subjected to western blotting with the indicated antibodies. B) Parental and MLK4 -/- HCT116 cells were stimulated with EGF (2nM-20 nM) at 37 {degree sign}C for 10 minutes. Cell lysates were subjected to western blotting with the indicated antibodies.

PDF file - 47K, List of tumors and number of samples for each histological type used for the mutational profile of MLK4 (GIST: Gastrointestinal Stromal tumors)

PDF file - 246K, A) Proliferation rates of HCT116 parental and MLK4 -/- cancer cells (two independent clones).*p value < 0.05; **p value < 0.01; error bars represent s.d; FC, fold change. B) Expression levels of the MLK4 protein were assessed by western blotting in control (ctrl) and MLK4 knock down LoVo cells (upper panel). C) Proliferation rates of ctrl and MLK4 knock down LoVo cells grown in plastic . *p value < 0.05; **p value < 0.01; error bars represent s.d; FC, fold change. D) Proliferation rates of Ctrl and MLK4 knock down LoVo cells grown in low adherence conditions. *p value < 0.05; **p value < 0.01; error bars represent s.d; FC, fold change. E) Anchorage-independent growth (soft agar) assay performed on control and MLK4 knock down LoVo cells. ***p value < 0.001; error bars represent s.d.

PDF file - 30K, Primers used for PCR amplification and sequencing of the MLK4 exons in tumor samples.

PDF file - 211K, A) Tumors formed by A549 cancer cells expressing wild type or two MLK4 mutants in xenograft mouse models. Cells were injected in the side of nude mice and tumor growth was measured at the indicated time points. Error bars represent s.e.m. B) Upon sacrifice the lungs were labeled by airway perfusion with the India ink, and superficial metastases were counted as described in panel C.*p value < 0.05; **p value < 0.01; error bars represent s.e.m.

Understanding the role of single-nucleotide polymorphisms (SNPs) in the pathological process represents a unique experimental challenge especially when the variants occur outside of coding regions. The noncoding SNP rs61764370 located in the 3-untranslated region of Kirsten rat sarcoma viral oncogene homolog (KRAS) has been implicated as a risk factor for the development of cancer and the response to targeted therapies. This cancer-associated variant is thought to affect the binding of the microRNA let-7, which allegedly modulates KRAS expression. Using site-specific homologous recombination, we inserted the rs61764370:T>GKRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. We observed a significant increase in cellular proliferation, as well as a reduction in the levels of the microRNA let-7a, let-7b, and let-7c. Transcriptional and biochemical analysis showed no concomitant change in the KRAS protein expression or modulation of the downstream mitogen activated kinase or PI3K/AKT signaling. These results suggest that the cancer-associated rs61764370 variant exerts a biological effect not through transcriptional modulation of KRAS but rather by tuning the expression of the microRNA let-7. (C) 2013 Wiley Periodicals, Inc.

Abstract Colorectal cancers (CRC) are commonly classified into those with microsatellite instability and those that are microsatellite stable (MSS) but chromosomally unstable. The latter are characterized by poor prognosis and remain largely intractable at the metastatic stage. Comprehensive mutational analyses have revealed that the mixed lineage kinase 4 (MLK4) protein kinase is frequently mutated in MSS CRC with approximately 50% of the mutations occurring in KRAS- or BRAF-mutant tumors. This kinase has not been characterized previously and the relevance of MLK4 somatic mutations in oncogenesis has not been established. We report that MLK4-mutated alleles in CRC are constitutively active and increase the transformation and tumorigenic capacity of RAS-mutated cell lines. Gene expression silencing or targeted knockout of MLK4 impairs the oncogenic properties of KRAS- and BRAF-mutant cancer cells both in vitro and in xenograft models. In establishing the role of MLK4 in intracellular signaling, we show it directly phosphorylates MEK1 (MAP2K1) and that MEK/ERK (MAPK1) signaling is impaired in MLK4 knockout cells. These findings suggest that MLK4 inhibitors may be efficacious in KRAS- and BRAF-mutated CRCs and may provide a new opportunity for targeting such recalcitrant tumors. Cancer Res; 73(6); 1912–21. ©2012 AACR.