Human Colon Cancer Tissues Research Articles

Role of cyclophilin B in tumorigenesis and cisplatin resistance in hepatocellular carcinoma in humans

Cyclophilin B (CypB) performs diverse roles in living cells, but its role in hepatocellular carcinoma (HCC) is largely unclear. To reveal its role in HCC, we investigated the induction of CypB under hypoxia and its functions in tumor cells in vitro and in vivo. Here, we demonstrated that hypoxia-inducible factor 1α (HIF-1α) induces CypB under hypoxia. Interestingly, CypB protected tumor cells, even p53-defective HCC cells, against hypoxia- and cisplatin-induced apoptosis. Furthermore, it regulated the effects of HIF-1α, including those in angiogenesis and glucose metabolism, via a positive feedback loop with HIF-1α. The tumorigenic and chemoresistant effects of CypB were confirmed in vivo using a xenograft model. Finally, we showed that CypB is overexpressed in 78% and 91% of the human HCC and colon cancer tissues, respectively, and its overexpression in these cancers reduced patient survival. These results indicate that CypB induced by hypoxia stimulates the survival of HCC via a positive feedback loop with HIF-1α, indicating that CypB is a novel candidate target for developing chemotherapeutic agents against HCC and colon cancer.

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin-focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3β pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src-miR-542-3p-ILK-FAK circuit plays a crucial role in controlling tumor progression.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.

A phase I study of NPC-1C, a novel therapeutic antibody to treat pancreas and colorectal cancers.

TPS141 Background: NPC-1C (N: ensituximab) is a chimeric monoclonal antibody being developed as a novel agent for pancreatic and colorectal cancers. This antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. N recognizes a unique immunogenic epitope with homology to a region of MUC5AC. This epitope is expressed by colon and pancreatic tumor tissues and cell lines. N does not cross-react with normal tissues (or MUC5AC from other types of cancers), except for occasional, weak binding to certain GI tract tissues. The N antibody shows ADCC activity against human colon and pancreatic tumor cells, but not control tumor cell lines. Human pancreatic tumor xenograft models showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. Several examples of human tumor tissues stained with N showed a strong correlation of N staining against pancreatic and colon tumors (45% of tumors strongly positive). Methods: A Phase I open label, multi-center dose escalation clinical trial with N is currently accruing patients with advanced, refractory pancreatic and colorectal cancer. A standard 3+3 design with an expansion cohort of 10 pancreatic cancer patients is planned. Patients will be treated with N starting at 1 mg/kg IV q2wks; cohort 1 has been completed without DLT and cohort 2 is being accrued; patients are hospitalized for 48 hours for observation after the first dose of N. Eligibility criteria included good end organ function and performance status, >20% of tumor tissue staining positive for MUC5AC, and an exclusion for QTc prolongation or history of hemolytic episodes. The primary objectives of the trial are to determine the safety/tolerability of escalating doses of N therapy and to assess pharmacokinetics and immune responses to the antibody at each dose level. Secondary objectives are to evaluate evidence of clinical benefit, as measured by RECIST criteria, and to explore the immunologic correlates associated with administration of N therapy. Results from this trial will determine the dosage levels to be used in further trials. NCT01040000.

Abstract 4079: Pilot study of copper chaperon ATOX1 in oncogenesis of colon cancer

Abstract INTRODUCTION: Copper is an essential element for normal function of many cellular enzymes. Copper homeostasis is maintained by a delicate network of copper transporters, which include human copper transporter 1(hCtr1), ATP7A, ATP7B, and copper chaperons including ATOX1, CCS, and Cox17. It has been known that copper is required for cell proliferation and tumor angiogenesis. Human tumor xenografts in mice with increased uptake of radioactive copper could be localized with positron emission tomography. Copper hypermetabolism might play a role in oncogenesis of human cancers. Recently, it was reported that ATOX1 is a transcription factor involved in copper-stimulated cell proliferation. In this pilot study, we analyzed expression of ATOX in human colon cancer tissues compared with its expression in normal colon tissues. In addition, preliminary experiments were conducted to study potential role of ATOX1 in oncogenesis of colon cancer. METHODS: Immunohistochemistry analysis was conducted to compare expression of ATOX1 in colon cancer tissues and normal colon tissues. A lentiviral vector encoding short-hairpin RNA (shRNA) specific for ATOX1 (Lenti-ATOX-shRNA) was constructed and used to transduce HCT-116 human colon cancer cells. Suppression of ATOX1 in HCT-116 cells transduced with Lenti-ATOX-shRNA (ATOX1-shRNA-HCT116) was confirmed by Western blot. MTT assay was conducted to assess effect of ATOX1 silencing on proliferation and sensitivity of ATOX1-shRNA-HCT-116 cells to copper, cisplatin and copper 8-hydroxyquinoline-2-carboxaldehyde thiosemicarbazide complex (CuHQTS), a new anticancer copper complex. The HCT-116 cells transduced by a lentivirus encoding a scramble shRNA (Scramble-shRNA-HCT116) were used as control. RESULTS: Over-expression of ATOX1 is identified in all of human colon cancer tissue samples (N=10), compared with expression of ATOX1 in normal colon tissue samples (N=10). Suppression of ATOX1 with ATOX1-shRNA in HCT116 cells was confirmed by Western blot assay. However, the results of MTT assay revealed no significant effect of ATOX1 silencing on proliferation and sensitivity of ATOX1-shRNA HCT116 cells to copper chloride, cisplatin, and CuHQTS, compared with proliferation and sensitivity of Scramble-shRNA-HCT116 to these drugs. CONCLUSIONS: ATOX1 holds potential as a new biomarker of colon cancer. Additional studies are necessary to determine the role of ATOX1 and other copper transporters in oncogenesis of colon cancer. Key words: colon cancer, copper metabolism, copper chaperon, ATOX1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2011-4079

Abstract 4582: Characterization of a new antibody that reacts with a tumor-specific variant of CEACAM-5 and CEACAM-6

Abstract Introduction: h16C3 is a humanized monoclonal antibody being developed as a novel biological treatment for several types of solid tumor. By mass spectroscopy identification and target validation research, we determined that h16C3 appears to recognize a variant form of CEACAM-5 and CEACAM-6, molecules with a well-known association to cancer. Here we show the pre-clinical activities of this new mAb which shows great potential as a therapeutic agent to treat solid tumors. Methods: The 16C3 antibody was selected from hundreds of hybridoma clones generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the potential of h16C3 as a therapeutic modality for solid tumors that express the variant CEACAM antigens. Results: Here we show that unlike most modalities targeting the CEACAM molecules, h16C3 reacts with variant CEACAM-5/6 expressed specifically by human tumor tissues as follows: 98% of human colorectal tumor tissues, 80% of pancreatic tumor tissues, 76% of lung tumor tissues, 43% of uterus tumor tissues, and 29% of breast tumor tissues stained positively with h16C3. Importantly, h16C3 did not cross-react significantly with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. We showed further that the h16C3 antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against antigen-negative control tumor cell lines. In vivo, the anti-tumor efficacy of h16C3 was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant anti-tumor action compared to control treated mice, including some complete tumor regressions. The bio-distribution of radiolabeled h16C3 in mice with pre-established human tumors exhibited specific accumulation of h16C3 at the tumor site, with little or no binding or accumulation in several major organ systems. Conclusions: The pre-clinical studies of h16C3 to date indicate that it has significant, specific anti-tumor potency, and suggest that it may have clinical activity in cancer patients whose tumor expresses the variant CEACAM-5/6 epitope. The therapeutic humanized 16C3 antibody is being developed for testing in a Phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4582. doi:10.1158/1538-7445.AM2011-4582

Abstract 4584: Pre-clinical studies of a novel antibody to treat pancreatic and colorectal cancers

Abstract Introduction: NPC-1C is a chimeric monoclonal antibody being developed as a novel biological treatment for pancreatic and colorectal cancers. NPC-1C appears to recognize a variant form of MUC5AC expressed specifically by human pancreatic and colorectal tumor tissues and cell lines. Here we show pre-clinical studies in preparation for testing this therapeutic for a Phase I clinical trial. Methods: The NPC-1C antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the GMP-grade antibody. Results: Here we show that NPC-1C reacts with colorectal and pancreatic tumor tissues, but does not cross-react with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. The NPC-1C antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against control tumor cell lines. In vivo, the anti-tumor efficacy of NPC-1C was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. The pre-clinical toxicity profile of NPC-1C in normal BALB/c mice demonstrated little, if any, toxicity following single or multiple intravenous injections. The bio-distribution of radiolabeled NPC-1C in mice with pre-established human tumors revealed specific accumulation of NPC-1C at the tumor site, with little or no binding or accumulation in several major organ systems. Finally, the cytokine and antibody responses to NPC-1C were studied in the pre-clinical mouse model. Although anti-NPC-1C antibodies were detected (as expected) in a time-dependent fashion, there were no changes detect in Type I and Type II cytokines either 3 days or 14 days after intravenous administration. Conclusions: The pre-clinical development of NPC-1C indicates that it is safe and efficacious, and suggests that it may have clinical activity in patients whose tumor expresses the variant MUC5AC epitope. The potential clinical application for this antibody was bolstered by showing approximately 50-70% of human pancreatic and colon tumor tissues stained positively with NPC-1C. The therapeutic antibody is now being tested in a Phase I clinical trial in patients with advanced pancreatic and colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4584. doi:10.1158/1538-7445.AM2011-4584

Clinicopathological correlation of beclin-1 expression in human colon cancer.

464 Background: We investigated the expression of beclin-1 and bcl-2 in human colon cancer tissue by immunohistochemical analysis using a tissue microarray in relation to other prognostic factors. Methods: Tissue samples from 78 cases of invasive colon cancer were used for the present study. A positive control for bcl-2 expression was small lymphocytes in the mantle zone. Immunohistochemical staining for beclin-1 and bcl-2 was evaluated according to intensity and proportion. The intensity score was determined as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The proportion score was determined as 1 (< 30% of tumor cells) and 2 (≥ 30% of tumor cells). The intensity score and proportion score were multiplied together for a total score. Total scores were as follows: 0-1 (negative) and 2-6 (positive). Results: The normal colonic epithelium showed weak to no beclin-1 expression. Positive beclin-1 expression and positive bcl-2 expression of colon cancer cells was observed in 48 cases (61.5%) and 10 cases (12.8%), respectively. beclin-1 expression was significantly correlated with lymph node metastasis (p = 0.035) Bcl-2 expression in colon cancer cells showed no significant correlation with clinocopathologic factors studied. Beclin-1 expression was not correlated with bcl-2 expression in colon cancer cells (p = 0.348). Conclusions: In conclusion, we have characterized the expression of beclin-1 in human colon cancer and the relationship with bcl-2 protein. Our results suggest that it seems reasonable to assume that beclin-1 might play a role in the development of colorectal cancer. No significant financial relationships to disclose.

NPC-1C: A novel, therapeutic antibody to treat pancreas and colorectal cancers.

235 Background: NPC-1C (ensituximab) is a chimeric monoclonal antibody being developed as a novel biologic treatment for pancreatic and colorectal cancers. This antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. The NPC-1C epitope appears to be expressed specifically by human colon and pancreatic tumor tissues and cell lines. Methods: Antitumor activity was established in vitro by measuring ADCC with a standard 4-hour 111-Indium release assay on pancreatic and colorectal cancer cell lines. In vivo antitumor efficacy of NPC-1C was tested using pre-established subcutaneous human pancreatic tumor xenograft models. Results: In vitro, the NPC-1C antibody exhibits ADCC activity specifically against human colon and pancreatic tumor cells, but not against control tumor cell lines. The in vivo data showed significant, and reproducible, antitumor action, including some complete tumor regressions. The clinical application for this antibody was bolstered by several examples of human tumor tissues stained with biotin-conjugated NPC-1C that showed a strong correlation of NPC-1C staining against pancreatic and colon tumors. Approximately 45% of tumors stained strongly positive. The staining pattern was typical of elaborated mucin expression, but also showed cytoplasmic and cell membrane staining. Conclusions: A phase I open label, multicenter dose escalation clinical trial with NPC-1C is currently accruing patients with advanced pancreatic and colorectal cancer who are refractory to standard therapy. The primary objectives of the phase I clinical trial are to determine the safety and tolerability of escalating doses of NPC-1C monoclonal antibody therapy and to assess pharmacokinetics and select immune responses to the antibody at each dose level. Secondary objectives are to evaluate evidence of clinical benefit and to explore the immunologic correlates associated with administration of NPC-1C. Results from this trial will determine the minimum standard dosage levels to be used in further trials. [Table: see text]

In vitro and in vivo activities of KRN330, a fully human monoclonal antibody against colon cancer.

432 Background: KRN330 is a novel recombinant human IgG1 monoclonal antibody (mAb) targeting A33 surface differentiation antigen that is uniformly expressed on the surface of 95% of colorectal cancer (CRC) cells. In this study, we characterized the activity of KRN330 for its in vitro properties, as well as for its in vivo antitumor activity. Methods: A kinetic analysis of the interaction between KRN330 and recombinant human A33 was conducted using a Biacore 3000. Western blot analysis was conducted using A33 expressing COLO205 lysates under reducing and non-reducing conditions. Binding of KRN330 to human colorectal cancer tissues were investigated using FITC-labeled KRN330. We also developed more conventional staining methods of A33 and investigated A33 expression using human colon cancer tissue microarray (TMA). ADCC and CDC activities of KRN330 were assessed using a standard 51Cr release assay. A33 expression levels of 14 CRC cell lines were analyzed using flow cytometer. In vivo antitumor activities of KRN330 alone or in combination with chemotherapeutic agents against subcutaneous or intraperiotoneal human CRC (COLO205 and LS174T) models were investigated using mice and rats xenograft model. Results: A kinetic analysis revealed that KRN330 showed a high binding affinity to A33. Western blot analysis also showed that antibody recognized not any protein under reducing condition, but non-reducing condition. A33 staining of TMA with 204 different samples revealed the majority of tumor expressed A33. KRN330 exhibited ADCC activity against A33 expressing human colorectal cancer cell lines which include both K-ras wild and mutated types. KRN330 showed dose-dependent antitumor activities in vivo. KRN330 also significantly prolonged survival of human colon tumor bearing mice. In addition, combination treatment of KRN330 with irinotecan showed increased antitumor activitiy and prolongation of survival, compared to either irinotecan or KRN330 alone. Conclusions: These results suggest that KRN330 is a promising candidate of novel therapy for CRC. The phase I/II study of KRN330 plus irinotecan in patients with second line metastatic CRC is ongoing. [Table: see text]

O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy

O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Although recent studies have shown that O-GlcNAcylation plays essential roles in breast cancer progression, it is also necessary to know whether O-GlcNAcylation is involved in other types of human cancer. In this study, O-GlcNAcylation levels and the expressions of OGT and OGA in human lung and colon cancer tissues were examined by immunohistochemistry analysis. We found that O-GlcNAcylation as well as OGT expression was significantly elevated in the cancer tissues compared with that in the corresponding adjacent tissues. Additionally, the roles of O-GlcNAcylation in the malignancy of lung and colon cancer were investigated in vitro. The results showed that O-GlcNAcylation markedly enhanced the anchorage-independent growth of lung and colon cancer cells; O-GlcNAcylation could also enhance lung and colon cancer invasion in a context-dependent manner. All together, this study suggests that O-GlcNAcylation might play important roles in lung and colon cancer formation and progression, and may be a valuable target for diagnosis and therapy of cancer.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133(hi) and CD133⁻ cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133⁻ cells showed that CD133 was highly expressed in colorectal cancer cell lines. Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.

基于表面增强拉曼光谱的结肠癌组织免疫分析

基于表面增强拉曼光谱的结肠癌组织免疫分析

Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition

BackgroundActivating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer.MethodsRegulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression.ResultsThe results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer.ConclusionIn conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.

Involvement of ST6Gal I in the biosynthesis of a unique human colon cancer biomarker candidate, α2,6-sialylated blood group type 2H (ST2H) antigen

The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

PTHrP promotes colon cancer cell migration and invasion in an integrin α6β4-dependent manner through activation of Rac1

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Rac1 GTPase enhances colon cancer cell migration and invasion. Here we report a positive correlation between PTHrP expression and Rac1 activity in LoVo (human colon cancer) cells. The positive effects of PTHrP on Rac1 activity and on cell migration and invasion are mediated via the guanine nucleotide exchange factor Tiam1. Knockdown of integrin α6β4, which is upregulated by PTHrP, negates the PTHrP-mediated increase in Rac1 activation. Integrin α6β4 signals synergistically with growth factor receptors to activate the phosphatidylinositol 3-kinase (PI3-K) pathway. Chemical inhibition of PI3-K negates the PTHrP-mediated effects on Tiam1 and Rac1 activity. Tumors from PTHrP-overexpressing LoVo cells also show increased expression of Tiam1. Taken together, these observations provide evidence of a link between PTHrP and Rac1 activity through integrin α6β4, resulting in enhanced cell migration and invasion. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.

Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes

Detection of somatic mutations in clinical cancer specimens is often hampered by excess wild-type DNA. The aim of this study was to develop a simple and economical protocol without using fluorescent probes to detect low-level mutations. In this study, we combined peptide nucleic acid (PNA)-clamping PCR with asymmetric primers and a melting curve analysis using an unlabeled detection probe. PNA-clamping PCR, which suppressed amplification of the wild-type allele, was more sensitive for KRAS codon 12 mutation detection than nonclamping PCR in 5 different mutant cell lines. Three detection probes were tested (a perfectly matched antisense, a mismatched antisense, and a mismatched sense), and the mismatched sense detection probe showed the highest sensitivity (0.1% mutant detection) under clamping conditions. With this probe, we were able to detect not only the perfectly matched KRAS mutation, but also 4 other mismatched mutations of KRAS. We then applied this protocol to 10 human colon cancer tissues with KRAS codon 12 mutations, successfully detecting the mutations in all of them. Our data indicate that the combination of perfectly matched antisense PNA and a mismatched sense detection probe can detect KRAS mutations with a high sensitivity in both cell lines and human tissues. Moreover, this study might prove an easily applicable protocol for the detection of low-level mutations in other cancer genes.

Discrimination of Patients with Microsatellite Instability Colon Cancer using 1H HR MAS MR Spectroscopy and Chemometric Analysis

The primary aim of this study was to analyze human colon cancer and normal adjacent tissue using (1)H HR MAS MR spectroscopy and chemometric analyses, evaluating possible biomarkers for colon cancer. The secondary aim was to investigate metabolic profiles of tissue samples (n = 63, 31 patients) with microsatellite instability (MSI-H) compared to microsatellite stable (MSS) colon tissue. Our hypothesis was that this method may provide an alternative to MSI genotyping. Cancer samples were clearly separated from normal adjacent mucosa by 100% accuracy. Several metabolites such as lactate, taurine, glycine, myo-inositol, scyllo-inositol, phosphocholine (PC), glycerophosphocholine (GPC), creatine, and glucose were identified as potential biomarkers for cancer detection. Adenomas (n = 4) were also separated from cancer and normal samples mainly based on higher GPC and PC levels. Interestingly, metabolic differences in normal colon mucosa between MSI-H and MSS patients were observed. MSI status was validated with 80% accuracy with a sensitivity and specificity of 79% and 82%, respectively, including both cancer and normal samples The metabolic differences between MSI-H and MSS may be very interesting in the early detection of cancer development and of high clinical importance in the work of improving diagnosis and characterization of colon cancer.

Abstract LB-161: Colospheres: 3D ex vivo microtumors with more aggressive phenotype than original human colon cancer tissues

Abstract The ex vivo colospheres are a newly characterised three-dimensional colon cancer multicellular model, derived from mechanically dissociated human colon cancer tissue (Br J Cancer 2009, 101:473-82). We have previously demonstrated that this short term culture model is exclusively formed by cancer cells and associated with tumor aggressiveness. To further investigate the potential interest of colospheres as micrometastasis or cancer stem cell model, we used 3 human colon cancer xenografts directly established from 3 patient colon adenocarcinoma in Nude mice (Cancer Res 2007, 67:398-407). These xenograft tissues are able to give rise to numerous colospheres in only 3 days after mechanical dissociation. This approach allows working with reproducible biological tumor material, in which all human part is known to be cancer cells. Xenograft tissues and derived colospheres were collected for gene expression study. Expression of 78 genes, involved in stemness, proliferation, epithelial-to-mesenchymal transition and metastasis, has been studied using real-time RT-PCR. All the probes used are human specific and do not cross with mouse genome. Consequently, the comparison of gene expression profiles corresponds to the comparison of gene expression in colosphere-forming cells versus that in the cancer cell counterpart from xenograft tissue. Gene expression clustering analysis clearly showed that for the 3 colon adenocarcinoma, colospheres matched with their parent xenografts. This first point is important because it demonstrated 1) the lack of ex vivo culture artefact (which would have led to classification into 2 groups: all xenografts opposed to all colospheres); 2) the relevance of colospheres for mimicking in vivo tumor cells. Nevertheless, in all 3 pairs xenograft/colospheres, 3 genes were found overexpressed: ALDH1, KLF4 and PLAUR. This overexpression has to be put in line with high tumorigenicity of these colospheres when injected into mice (Br J Cancer 2009, 101:473-82). Gene expression increase will be confirmed at protein level. To gain also information about location of the cells expressing ALDH1, KLF4 and PLAUR, we developed an immunostaining protocol for confocal microscopy and in situ protein detection (BMC Cancer, in revision). In conclusion, these preliminary data underline the interest of colospheres as ex vivo microtumor model with cancer initiating-cell functions. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-161.