Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Abstract

Abstract MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 463. doi:1538-7445.AM2012-463