SUMMARY The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer.
Oncogenic mutations in the K-ras gene occur in Ϸ50% of human colorectal cancers. However, the precise role that K-ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-ras V12 allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-ras V12 does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf͞MEK͞ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-ras V12 expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic Kras V12 allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf͞MEK͞ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-ras oncogene. However, activation of K-ras V12 after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.colorectal cancer ͉ renal carcinoma ͉ Wnt signaling
Engagement of the T cell antigen receptor (TcR)1 with the antigen-major histocompatibility complex on antigen-presenting cells triggers a complex TcR signaling cascade that leads to T cell activation and cytokine secretion (1). During this process, T cells express the autocrine growth factor interleukin 2 (IL-2), which promotes T cell proliferation by interacting with the IL-2 receptor, which is also up-regulated on activated T cells. The transcriptional regulation of the IL-2 gene has been extensively analyzed at the IL-2 promoter, a 275-bp region located upstream of the transcriptional start site of the gene (2, 3). Several transcription factors have been identified to bind elements within this regulatory region, including AP-1, NF-B, and the nuclear factor of activated T cells (NFAT) (2).The transcription factor NFAT plays an essential role in IL-2 expression. Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-3, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor ␣, granulocyte-macrophage colony-stimulating factor, and ␥-IFN (4, 5). NFAT is a complex composed of a cytoplasmic subunit and an inducible nuclear component comprised of AP-1 (Fos/ Jun) family members. At least four structurally related NFAT cytoplasmic subunit members, NFATp/NFAT1, NFATc/ NFAT2, NFAT3, and NFATX/NFATc3/NFAT4, have been identified (5). NFAT proteins share a conserved domain located toward the C terminus (6) that binds DNA and also participates in cooperative protein-protein interactions with AP-1 transcription factors (7,8). Immediately N-terminal to the DNA-binding domain is a second conserved module of ϳ300 residues known as the NFAT homology (NFAT-h) region. The N terminus of NFAT, including the NFAT-h region, regulates nuclear/cytoplasm trafficking in response to changes in intracellular Ca 2ϩ concentrations. In resting T cells, the protein is retained in the cytoplasm and its NFAT-h domain is heavily phosphorylated. Engagement of the TcR or treatment of cells with the Ca 2ϩ ionophore activates the Ca 2ϩ /calmodulin-dependent Ser/Thr phosphatase, calcineurin. CaN dephosphorylates the NFAT-h domain, resulting in translocation of NFAT to the nucleus (9).
Pleomorphic rhabdomyosarcoma is the most common variant of this tumour in adults and has a very poor outcome. Two genes which are known to play a role in rhabdomyosarcoma development are KRas and p53. In the majority of human tumours, p53 abnormalities are point mutations that result in the expression of a mutant form of the protein. It is now hypothesized that these mutant forms of p53 may be playing an oncogenic role, over and above simple loss of the wild-type function. In this study, we use Cre-LoxP technology to develop a novel mouse model of rhabdomyosarcoma, crossing mice expressing a common KRas mutation (G12V) with mice that either lose p53 expression or express a mutant form of p53. We use this model to explore the different effects of p53 loss and mutation in the setting of an activating KRas mutation. We found that either complete loss of p53 (p53(fl/fl)) or the expression of one mutant p53 allele with concomitant loss of the second allele (p53(R172H/+)) resulted in the rapid development of rhabdomyosarcoma in 15/16 and 19/19 mice, respectively. In contrast, there was a marked difference between mice which lose a single copy of p53 (p53(fl/+)) and mice expressing a single copy of mutant p53 (p53(172H/+)). Fourteen out of 16 p53(R172H/) mice developed rhabdomyosarcoma, compared with two out of 31 p53(fl/+) mice. As a consequence of this, p53(fl/+) mice had a median lifespan nearly double that of the p53(R172H/+) mice. To underline the enhanced effect of p53 mutation in tumour progression, metastases were seen only in those mice which expressed the mutant form. These data demonstrate that mutant p53 can co-operate with activated, mutant KRas to influence tumourigenesis and metastatic potential, over and above simple loss of normal protein function.
Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear β-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors β-catenin and c-Myc. β-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being β-catenin-dependent but c-Myc-independent in the liver.
The Adenomatous polyposis coli (Apc) gene is mutated in up to 80% of sporadic colorectal cancers. After Apc loss, there is deregulation of the Wnt signaling pathway and transactivation of T-cell factor/leukemia enhancing factor target genes such as C-Myc. This review focuses on recent data highlighting the importance of the C-Myc oncogene and its transcriptional targets in establishing all of the phenotypes caused by the deletion of the Apc tumor suppressor gene within the intestinal epithelium. The importance of investigating Apc and C-Myc gene function in the correct tissue context is also discussed. [Cancer Res 2008;68(13):4963-6]
In order to explore the distribution of hormone-responsive cells in skeletal tissues, we have examined the effects of synthetic bovine parathyroid hormone N-terminal peptide (bPTH 1-34) and salmon calcitonin (sCT) on cyclic AMP levels in periosteum-free rat calvaria, segments of periosteum, and in isolated cells dispersed from each tissue by collagenase digestion. Synthetic bovine PTH increased cyclic AMP levels to a greater degree in calvaria and in isolated bone cells than in the periosteal segments and cells, whereas sCT was more effective in the periosteal than in the bone systems. Primary cultures prepared from bone and periosteal cell populations exhibited progressive increases in their responsiveness to bPTH (1-34) and progressive decreases in responsiveness to sCT. After six days in the culture, bone cells failed to respond to sCT, and sCT did not modify their response simultaneously added bPTH (1-34). Six-day periosteal cell cultures exhibited residual sCT responsivity and an additive response upon simultaneous exposure to high concentrations of bPTH (1-34) and sCT suggesting separate sites of hormone action. Adenosine, a known stimulator of bone cell adenylyl cyclase, caused a greater increase in periosteal cell than in bone cell cyclic AMP. bPTH (1-34)-responsive cells which enrich periosteum-free bone may be osteoblasts, in view of their histological prominence in this tissue and in the bone cell isolates. Periosteal cells which responded to sCT and to adenosine preferentially are unidentified. Although periosteal segments contained numerous fibroblast-like cells, skin fibroblasts cultured from the same fetuses were sCT-insensitive. Growth in primary culture appears to alter the number of hormone-responsive cells or responsiveness of existing cells to each hormone, or both.
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