Erik Bruyneel

The role of bile acids in carcinogenesis

Bile acids play a role in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. Some bile acids stimulate growth of normal colonic and adenoma cells, but not of colorectal cancer cells. Moreover, bile acids stimulate invasion of colorectal cancer cells, at least in vitro. One possible mechanism of action is bile acid-induced DNA binding and transactivation of the activator protein-1 (AP-1) by co-operate activation of extracellular signal-regulated kinases (ERKs) and PKC signaling. In the present paper, we review the mechanisms by which bile acids influence carcinogenesis.

Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells

Trefoil factors (TFFs) are protease‐resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose‐response curves in src‐transformed MDCKts.src and PCmsrc cells (EC50 = 20–40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src‐pS2 cells and human colonic HCT8/S11‐pS2 cells established from a sporadic tumor. Furthermore, we found that TFF‐mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3′‐kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed byWestern blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.—Emami, S., Le Floch, N., Bruyneel, E., Thim, L., May, F., Westley, B., Rio, M.‐C., Mareel, M., Gespach, C. Induction of scattering and cellular invasion by trefoil peptides in src‐ and RhoA‐transformed kidney and colonic epithelial cells. FASEB J. 15, 351‐361 (2001)

Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis

Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho–Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways

We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX‐1 and COX‐2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U‐73122), cyclooxygenases (SC‐560, NS‐398), and the thromboxane A2 receptor (TXA2‐R) antagonist SQ‐295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2‐R mimetic U‐46619, constitutively activated forms of the heterotrimeric G‐proteins Gαq (AGαq), Gα12, Gα13 (AGα12/13), which are signaling elements downstream of TXA2‐R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src‐pS2 cells and human colorectal cancer cells HCT8/S11‐pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2‐R dependent. We detected a marked induction of COX‐2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2‐R‐dependent invasion pathway, which is monitored via a Rho‐ and Gα12/Gα13‐independent mechanism using the Gαq/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.—Rodrigues, S., Nguyen, Q.‐ D., Faivre, S., Bruyneel, E., Thim, L., Westley, B., May, F., Flatau, G., Mareel, M., Gespach, C., Emami, S. Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase‐ and thromboxane A2 receptor‐dependent signaling pathways. FASEB J. 15, 1517–1528 (2001)

Bile acids stimulate invasion and haptotaxis in human colorectal cancer cells through activation of multiple oncogenic signaling pathways.

Bile acids are implicated in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. We examined whether bile acids stimulate cellular invasion of human colorectal and dog kidney epithelial cells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidney MDCKT23 cells were seeded on top of collagen type I gels and invasive cells were counted after 24 h incubation. Activation of the Rac1 and RhoA small GTPases was investigated by pull-down assays. Haptotaxis was analysed with modified Boyden chambers. Lithocholic acid, chenodeoxycholic acid, cholic acid and deoxycholic acid stimulated cellular invasion of SRC- and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originating from a sporadic tumor, but were ineffective in premalignant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation of haptotaxis and was dependent on the RhoA/Rho-kinase pathway and signaling cascades using protein kinase C, mitogen-activated protein kinase, and cyclooxygenase-2. Accordingly, BA-induced invasion was associated with activation of the Rac1 and RhoA GTPases and expression of the farnesoid X receptor. We conclude that bile acids stimulate invasion and haptotaxis in colorectal cancer cells via several cancer invasion signaling pathways.

Monosialyl‐Gb5 organized with cSrc and FAK in GEM of human breast carcinoma MCF‐7 cells defines their invasive properties

Two human mammary carcinoma cell variants, MCF‐7/AZ and MCF‐7/6, show the same composition in their glycosphingolipid‐enriched microdomain (GEM) with regard to globo‐series structures Gb3, Gb4, Gb5, monosialyl‐Gb5, GM2, and cSrc and FAK. Both variants are non‐invasive into collagen gel layer, and showed similar motility in wound migration assay. Whereas invasiveness and motility of MCF‐7/AZ cells were enhanced greatly by treatment with mAb RM1 directed to monosialyl‐Gb5, the same RM1 treatment had no effect on MCF‐7/6. cSrc and FAK of MCF‐7/AZ, but not MCF‐7/6, were activated by RM1 treatment. Thus, malignancy of MCF‐7 is highly dependent on monosialyl‐Gb5, and its activation of cSrc and FAK in GEM.

Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells

TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma–carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.