Cyclooxygenase-2/carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells
Corresponding Author
Pasquale Sansone
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Pharmacology, University of Bologna, Bologna, Italy
Correspondence to: Pasquale SANSONE, Ph.D., Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy.E-mail: [email protected] or Massimiliano BONAFÉ, M.D., Department of Experimental Pathology, University of Bologna, Bologna, Italy.Tel.: +39-051-636-3902E-mail: [email protected]Search for more papers by this authorGiulia Piazzi
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
These authors contributed equally to the work.
Search for more papers by this authorPaola Paterini
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Experimental Evolutionary Biology, University of Bologna, Bologna, Italy
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
These authors contributed equally to the work.
Search for more papers by this authorAntonio Strillacci
Department of Experimental Evolutionary Biology, University of Bologna, Bologna, Italy
Search for more papers by this authorClaudio Ceccarelli
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
Search for more papers by this authorFrancesco Minni
Department of Surgical and Anesthesiological Sciences, University of Bologna, Bologna, Italy
Search for more papers by this authorGuido Biasco
Institute of Hematology and Medical Oncology, St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Search for more papers by this authorPasquale Chieco
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Search for more papers by this authorCorresponding Author
Massimiliano Bonafè
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Experimental Pathology, University of Bologna, Bologna, Italy
Correspondence to: Pasquale SANSONE, Ph.D., Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy.E-mail: [email protected] or Massimiliano BONAFÉ, M.D., Department of Experimental Pathology, University of Bologna, Bologna, Italy.Tel.: +39-051-636-3902E-mail: [email protected]Search for more papers by this authorCorresponding Author
Pasquale Sansone
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Pharmacology, University of Bologna, Bologna, Italy
Correspondence to: Pasquale SANSONE, Ph.D., Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy.E-mail: [email protected] or Massimiliano BONAFÉ, M.D., Department of Experimental Pathology, University of Bologna, Bologna, Italy.Tel.: +39-051-636-3902E-mail: [email protected]Search for more papers by this authorGiulia Piazzi
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
These authors contributed equally to the work.
Search for more papers by this authorPaola Paterini
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Experimental Evolutionary Biology, University of Bologna, Bologna, Italy
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
These authors contributed equally to the work.
Search for more papers by this authorAntonio Strillacci
Department of Experimental Evolutionary Biology, University of Bologna, Bologna, Italy
Search for more papers by this authorClaudio Ceccarelli
Institute of Hematology and Medical Oncology and Laboratory of Medicine, University of Bologna, Bologna, Italy
Search for more papers by this authorFrancesco Minni
Department of Surgical and Anesthesiological Sciences, University of Bologna, Bologna, Italy
Search for more papers by this authorGuido Biasco
Institute of Hematology and Medical Oncology, St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Search for more papers by this authorPasquale Chieco
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Search for more papers by this authorCorresponding Author
Massimiliano Bonafè
Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy
Department of Experimental Pathology, University of Bologna, Bologna, Italy
Correspondence to: Pasquale SANSONE, Ph.D., Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy.E-mail: [email protected] or Massimiliano BONAFÉ, M.D., Department of Experimental Pathology, University of Bologna, Bologna, Italy.Tel.: +39-051-636-3902E-mail: [email protected]Search for more papers by this authorAbstract
Inflammation promotes colorectal carcinogenesis. Tumour growth often generates a hypoxic environment in the inner tumour mass. We here report that, in colon cancer cells, the expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) associates with that of the hypoxia response gene carbonic anhydrase-IX (CA-IX). The COX-2 knockdown, achieved by the stable infection of a COX-2 specific short harpin RNA interference (shCOX-2), down-regulates CA-IX gene expression. In colorectal cancer (CRC) cells, PGE2, the main COX-2 gene products, promotes CA-IX gene expression by ERK1/2 activation. In normoxic environment, shCOX-2 infected/CA-IX siRNA transfected CRC cells show a reduced level of active metalloproteinase-2 (MMP-2) that associates with a decreased extracellular matrix invasion capacity. In presence of hypoxia, COX-2 gene expression and PGE2 production increase. The knockdown of COX-2/CA-IX blunts the survival capability of CRC cells in hypoxia. At a high cell density, a culture condition that creates a mild pericellular hypoxic environment, the expression of COX-2/CA-IX genes is increased and triggers the invasive potential of colon cancer cells. In human colon cancer tissues, COX-2/CA-IX protein expression levels, assessed by Western blot and immunohistochemistry, correlate each other and increase with tumour stage. In conclusion, these data indicate that COX-2/CA-IX interplay promotes the aggressive behaviour of CRC cells.
Supporting Information
Fig. S1. COX-2/CA-IX up-regulation sustains hypoxia survival in colorectal cancer cells. (A) HCT-116 cells transiently transfected with CTR/COX-2 siRNA (1 mg, 48 hrs pre-exposure) and HCA-7 cells stably infected with shCTR/COX-2 pSUPER.retro vector exposed to 100 mM DFX for 48 hrs: Cell death assay 48 hrs and RT-PCR analysis of CA-IX and COX-2 mRNA level. (B) HCT-116 cells pre-treated with PGE2 1 mM for 24 hrs and/or transiently transfected with CA-IX/SCR siRNA (1 mg, 24 hrs pre-exposure) and then exposed to DFX 100 mM for 48 hrs: Cell death assay 48 hrs and RT-PCR analysis of CA-IX mRNA level. Data are expressed as mean ± S.D. of three replicates (n = 3) (ANOVA test, §p < 0.001, Post Hoc test adjustment for multiple comparison was applied when required).
Fig. S2. COX-2/CA-IX expression in CRC tissues. Representative immunohistochemical staining of COX-2 and CA-IX protein level in stage I-III-IV. CRC cancer tissue samples (scale bar, 500 mm,100 mm, 20 mm). IHC photos represent both the mass of the tumour (M) and the tumour host interface (THI).
Table S1. Western blot antibodies and conditions. Legends: TBS-TB buffer (20 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20, 5% bovine serum albumin) TBS-TM buffer (20 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20, 5% non-fat dry milk).
Table S2. Primers sequences and conditions. Legends: COX-2: cyclooxygenase-2; CA-IX: carbonic Anhydrase-IX; b2m: B-2 microglobulin; C-MET: Hepatocyte Growth Factor receptor; VEGF: vascular endothelial growth factor; HO-1: Heme oxygenase-1; BCRP-1: Breast Cancer Resistance Protein-1.
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