Greg Cohen

Epidermal growth factor receptor signaling is up-regulated in human colonic aberrant crypt foci.

Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA >/=2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 +/- 1.7-fold), transforming growth factor-alpha (14.4 +/- 5.0-fold), heparin-binding EGF-like growth factor (4.5 +/- 1.4-fold), cyclin D1 (4.6 +/- 0.7-fold), and cyclooxygenase-2 (COX-2; 9.3 +/- 4.2-fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 +/- 1.2-fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 +/- 1.3-fold), phosphoactive ErbB2 (2.6 +/- 0.4-fold), phosphoactive extracellular signal-regulated kinase (3.7 +/- 1.1-fold), and cyclin D1 (3.4 +/- 0.8-fold; P < 0.05). Ras was activated in 46% of ACF (3.2 +/- 0.4-fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF.

Epidermal Growth Factor Receptor Signaling Is Required for Microadenoma Formation in the Mouse Azoxymethane Model of Colonic Carcinogenesis

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.

Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators

PKC-δ is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-δ slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-δ dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-δ using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-δ caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21Waf1, a cyclin-dependent kinase (cdk) inhibitor in PKC-δ transfectants compared with empty vector (EV) transfected cells, whereas the PKC-δ specific inhibitor rottlerin (3 μM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21Waf1 expression. Concomitantly, compared to EV control cells, PKC-δ upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-δ increased binding of cdk inhibitor p27Kip1 to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-δ plays in cell growth and cell cycle regulation, we knocked down PKC-δ using specific siRNA oligonucleotides. PKC-δ specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-δ protein by more than 80% in Caco-2 cells. Moreover, PKC-δ knockdown enhanced cell proliferation (∼1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression (∼1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-ζ was not changed by PKC-δ siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-δ transfectants, compared to EV cells, PKC-δ upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-δ specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-δ regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-δ that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-δ in colonic carcinogenesis.

Ursodeoxycholic Acid Suppresses Cox-2 Expression in Colon Cancer: Roles of Ras, p38, and CCAAT/Enhancer-Binding Protein

In the azoxymethane (AOM) model of experimental rodent colon cancer, cholic acid and its colonic metabolite deoxycholic acid (DCA) strongly promote tumorigenesis. In contrast, we showed that ursodeoxycholic acid (UDCA), a low abundance bile acid, inhibited AOM tumorigenesis. Dietary UDCA also blocked the development of tumors with activated Ras and suppressed cyclooxygenase-2 (Cox-2) upregulation in AOM tumors. In this study, we compared the effect of dietary supplementation with tumor-promoting cholic acid to chemopreventive UDCA on Cox-2 expression in AOM tumors. Cholic acid enhanced Cox-2 upregulation in AOM tumors, whereas UDCA inhibited this increase and concomitantly decreased CCAAT/enhancer binding protein β (C/EBPβ), a transcriptional regulator of Cox-2. In HCA-7 colon cancer cells, DCA activated Ras and increased C/EBPβ and Cox-2 by a mechanism requiring the mitogen-activated protein kinase p38. UDCA inhibited DCA-induced p38 activation and decreased C/EBPβ and Cox-2 upregulation. Using transient transfections, UDCA inhibited Cox-2 promoter and C/EBP reporter activation by DCA. Transfection with dominant-negative 17N-Ras abolished DCA-induced p38 activation and C/EBPβ and Cox-2 upregulation. Taken together, these studies have identified a transcriptional pathway regulating Cox-2 expression involving Ras, p38, and C/EBPβ that is inhibited by UDCA. These signal transducers are novel targets of UDCAs chemopreventive actions.

Gene Signature Distinguishes Patients with Chronic Ulcerative Colitis Harboring Remote Neoplastic Lesions

Background:Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer. The standard method of surveillance for neoplasia in UC by colonoscopy is invasive and can miss flat lesions. We sought to identify a gene expression signature in nondysplastic mucosa without active inflammation that could serve as a marker for remote neoplastic lesions. Methods:Gene expression was analyzed by complementary DNA microarray in 5 normal controls, 4 UC patients without dysplasia, and 11 UC patients harboring remote neoplasia. Common gene ontology pathways of significantly differentially expressed genes were identified. Expression of genes which were progressively and significantly upregulated from controls to UC without neoplasia, to UC with remote neoplasia were evaluated by real-time polymerase chain reaction. Several gene products were also examined by immunohistochemistry. Results:Four hundred and sixty-eight genes were significantly upregulated, and 541 genes were significantly downregulated in UC patients with neoplasia compared with UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly upregulated from controls to nondysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increased expression of S100A9 and REG1&agr; in UC-associated cancer and in nondysplastic tissue from UC patients harboring remote neoplasia compared with UC patients without neoplasia and controls. Conclusions:Gene expression changes occurring as a field effect in the distal colon of patients with chronic UC identify patients harboring remote neoplastic lesions. These markers may lead to a more accurate and less invasive method of detection of neoplasia in patients with inflammatory bowel disease.

Ursodeoxycholic acid inhibits ras mutations, wild type ras activation and cyclooxygenase-2 expression in colon cancer

K-ras mutations occur frequently in colon cancer and contribute to autonomous growth. In the azoxymethane (AOM) model of colon cancer, in addition to K-ras mutations, we have shown that wild-type (WT) Ras can be activated by upstream pathways, including, e.g., signaling by ErbB receptors. Tumors with mutant or activated WT Ras had increased cyclooxygenase-2 (Cox-2) expression. We have also shown that ursodeoxycholic acid (UDCA) prevented AOM-induced colon cancer and suppressed Cox-2 induction. In this study, we examined the role of Ras in Cox-2 inhibition by UDCA. Rats were fed AIN-76A chow alone, or supplemented with 0.4% UDCA, and received 20 mg/kg AOM i.p. weekly x 2 weeks. At 40 weeks, rats were sacrificed, and tumors were harvested. K-ras mutations were assessed by primer-mediated RFLP, allele-specific oligonucleotide hybridization, and direct DNA sequencing. Ras was immunoprecipitated and defined as activated if [Ras - GTP/(Ras - GTP + Ras - GDP)] was >3 SD above normal colonocytes. Cox-2 mRNA was determined by reverse transcription-PCR, and protein expression was assessed by Western blotting and immunostaining. In the AOM alone group, Ras was activated by mutations in 8 of 30 (27%) tumors, and WT Ras was activated in 7 of 30 (23%) tumors. UDCA significantly suppressed the incidence of tumors with mutant Ras (1 of 31, 3.2%; P < 0.05) and totally abolished the development of tumors with activated WT Ras (0 of 10; P < 0.05). In the AOM alone group, Cox-2 was up-regulated >50-fold in tumors with normal Ras activity and further enhanced in tumors with mutant or signaling-activated Ras. UDCA significantly inhibited Cox-2 protein and mRNA levels in tumors with normal Ras activity. In summary, we have shown for the first time that UDCA suppressed the development of tumors with Ras mutations and blocked activation of WT Ras. Furthermore, UDCA inhibited Cox-2 induction by Ras-dependent and -independent mechanisms.