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Tumor-derived cell lines are indispensable tools for understanding the contribution of activated signaling pathways to the cancer phenotype and for the design and testing of targeted signal therapies. In our study, we characterize 10 colorectal carcinoma cell lines for the presence of mutations in the wnt, Ras/MAPK, PI3K and p53 pathways. The mutational spectrum found in this panel of cell lines is similar to that detected in primary CRC, albeit with higher frequency of mutation in the β-catenin and B-Raf genes. We have monitored activation of the wnt and Ras/MAPK pathways in these cells and analyzed their sensitivity to selective signaling inhibitors. Using β-catenin subcellular distribution as a marker, we show that cells harboring APC mutations have low-level activated wnt signaling, which can be blocked by the extracellular wnt inhibitor DKK-1, suggesting autocrine activation of this pathway; proliferation of these cells is also blocked by DKK-1. In contrast, cells with β-catenin mutations are unresponsive to extracellular wnt inhibition. Constitutive phosphorylation of MAPK is present in the majority of the cell lines and correlates with B-Raf but not K-Ras mutations; correspondingly, the proliferation of cells harboring mutations in B-Raf, but not K-Ras, is exquisitely sensitive inhibition of the MAPK pathway. We find no correlation between PI3K mutation or loss of PTEN expression and increased sensitivity to PI3K inhibitors. Our study discloses clear-cut differences in responsiveness to signaling inhibitors between individual mutations within an activated signaling pathway and suggests likely targets for signal-directed therapy of colorectal carcinomas. © 2009 UICC
The progression of colorectal cancer from adenomas to carcinomas is characterized by a series of genetic changes, which lead to the activation of oncogenes and to the inactivation of tumor suppressor genes, perturbing the self-renewal, proliferative, adhesive and survival characteristics of the cells.1, 2 Colorectal cancers are characterized by mutations in 4 major pathways: wnt, Ras/MAPK, p53 and DNA repair.
The APC gene is a key regulator of the wnt pathway. APC mutations are detected early in the adenoma-carcinoma progression,3 are the hallmark of Familial Adenomatous Polyposis (FAP) and also occur with high frequency (up to 80%) in sporadic CRC. Mutations of the APC gene cluster in the central region of the protein and result in truncations, which delete most of the β-catenin binding sites, the axin-binding domain and the microtubule-binding regions. These deletions have been associated with activation of the canonical wnt signaling pathway and cytosolic/nuclear accumulation of β-catenin4 but may also play an important, β-catenin independent, role in the loss of cell polarity observed in many colorectal carcinomas.5 Underlying the relevance of the wnt pathway in the aetiogenesis of CRC, mutations of other components in the canonical wnt pathway also occur with significant frequency in primary CRC: β-catenin is mutated in 1–3%6, 7 and Axin-2 in 3–30% of CRC, with the highest frequency in MSI tumors.8, 9 The most frequent mutation of β-catenin found in CRC occurs at codon 45, a serine critical for priming phosphorylation, but mutations of the other phosphorylation sites also occur; these mutations abolish phosphorylation-mediated degradation of β-catenin leading to its cytosolic and nuclear accumulation.
Components of the Ras/MAPK and Ras/PI3K pathway are also frequently mutated in CRC. Mutations in the proto-oncogene K-Ras are present in ∼50% of colorectal cancers,10, 11 occurring with similar frequency in both familial and sporadic colorectal cancers.12 The proto-oncogene B-Raf, a serine/threonine kinase activated by Ras-GTP, is mutated in 10–15% of sporadic colorectal cancers.1, 13, 14 The most common B-Raf mutation, accounting for ∼90% of all B-Raf mutations in human cancers, occurs at codon 600, resulting in an amino acid change from the neutral valine (V) to the negatively charged amino acid glutamic acid (E) and in the constitutive activation of B-Raf.15 Although both K-Ras and B-Raf activate the MEK/MAPK pathway, K-Ras also directly activates the PI3K pathway. The enzyme PI3Kinase comprises a p110 catalytic subunit and a p85 regulatory subunit16 and mutations can occur in both subunits.17, 18 Mutations of PI3Kinase occur with relatively high frequency in CRC (2–30% [Refs.19 and20]) and can be coincident with K-Ras mutations.21 The PI3K pathway has been linked to tumor invasion and metastasis22 as well as to suppression of apoptosis.23
Finally, TP53, a gatekeeper gene that controls cell cycle arrest and apoptosis, is mutated in 20–40% of colorectal cancers.24, 25 Expression of functional p53 is important in the prevention of cancer development from precancerous lesions,26 and mutations of p53 frequently occur together with mutations of tumor-suppressor genes or of proto-oncogenes.24 Thus, in colorectal cancer, the 4 signaling pathways—wnt, Ras/MAPK, PI3K and p53—each contribute to important aspects of tumorigenesis and act in concert during tumor development.
Our increased understanding of the processes that lead to cancer development has shifted the focus of new cancer therapies to targeted interventions, which attempt to block selectively the pathways activated by the genetic mutations present in individual cancers.27, 28 Given the complexity of the genetic changes present in cancer, and the still incomplete knowledge of how different pathways interact to establish and maintain tumors, much of the development phase relies on “in vitro” model systems. Cell lines derived from primary or metastatic colorectal cancers can provide invaluable experimental tools for understanding the molecular events leading to tumor formation and for the manipulation of relevant signaling pathways. For these experimental systems to be meaningful, however, we need to assess whether the cell lines reflect the genetic characteristics of human CRC, whether the genetic mutations lead to “addiction” of the cells to constitutively active signaling pathways and whether the tumorigenic potential of the cells can be manipulated by interfering with the activated pathways.
CRC lines from the LIM series, which were established over a long period of time from primary colorectal cancers, have been previously characterized phenotypically29–31 and have been used extensively as models for colorectal cancer, with emphasis on the responsiveness to growth factors,32 epithelial-to-mesenchymal transition33 and induction of differentiation.34 We now report a detailed analysis of the mutations present in these cells, focusing on the pathways known to be altered in primary CRC. We have used available inhibitors to determine the role of signaling pathway activation in the proliferation and survival of the cells. Our results indicate that it is possible to pharmacologically revert inappropriate activation of signaling pathways driven by particular mutations, with concomitant reduction in the proliferation, survival and/or malignancy of the cells; however, not all the pathways mutated in CRC are responsive to specific signal therapeutics.