Radiation-Stimulated ERK1/2 and JNK1/2 Signaling can Promote Cell Cycle Progression in Human Colon Cancer Cells

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways and cell cycle progression following exposure to ionizing radiation is largely unknown. Loss of K-RAS D13 expression in parental HCT116 colorectal carcinoma cells blunted basal ERK1/2, AKT and JNK1/2 activity by ~70%. P38 activity was not detected. Deletion of the allele to express activated K-RAS nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells, but did not restore or alter basal JNK1/2 and p38 activity. In parental cells radiation (1 Gy) caused stronger ERK1/2 pathway activation compared to that of the PI3K/AKT pathway. In H-RAS V12 cells radiation caused stronger PI3K/AKT pathway activation compared to that of the ERK1/2 pathway. Radiation (1 Gy) promoted S phase entry in parental HCT116 cells within 24h, but not in either HCT116 cells lacking K-RAS D13 expression or in H-RAS V12 cells. In parental cells radiation-stimulated S phase entry correlated with ERK1/2-, JNK1/2- and PI3K-dependent increased expression of cyclin D1 and cyclin A, and to a lesser extent cyclin E, 6–24 h after exposure. Cyclin A and cyclin D1 expression were not increased by radiation in cells lacking K-RAS D13 expression or in H-RAS V12 cells. Radiation (1 Gy) modestly enhanced expression of p53, hMDM2 and p21 in parental cells 2-6h after exposure, which was abolished in cells lacking K-RAS D13 expression. Introduction of H-RAS V12 into cells lacking mutant active RAS partially restored radiation-induced expression of p21 and p53, and enhanced the induction of hMDM2 beyond that observed in parental cells. Collectively, our findings argue that the coordinated activation of multiple signaling pathways, in particular ERK1/2 and JNK1/2, by radiation is required to elevate the expression of G1 and S phase cyclin proteins and to promote S phase entry in human colon carcinoma cells expressing wild type p53. In HCT116 cells H-RAS V12 promotes hMDM2 expression after radiation exposure which correlates with reduced p53 expression and increased cell survival.     

Effects of activin and TGFβ on p21 in colon cancer

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21(cip1/waf1)). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.     

Oncogenic KRAS provides a uniquely powerful and variable oncogenic contribution among RAS family members in the colonic epithelium

Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS(G13D) signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context-dependent.     

The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-microM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 microM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21(Cip/WAF1) on adding 100 microM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21(Cip/WAF1) induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21(Cip/WAF1) inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.     

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.