Cross-Talk Between Phospho-STAT3 and Plcg1 Plays a Critical Role in Colorectal Tumorigenesis
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Published OnlineFirst August 12, 2011; DOI: 10.1158/1541-7786.MCR-11-0147 Molecular Cancer Signaling and Regulation Research Cross-talk between Phospho-STAT3 and PLCg1 Plays a Critical Role in Colorectal Tumorigenesis Peng Zhang1, Yiqing Zhao1,5, Xiaofeng Zhu2, David Sedwick3, Xiaodong Zhang6, and Zhenghe Wang1,4 Abstract Hyperphosphorylation at the Y705 residue of signal transducer and activator of transcription 3 (STAT3) is implicated in tumorigenesis of leukemia and some solid tumors. However, its role in the development of colorectal cancer is not well defined. To rigorously test the impact of this phosphorylation on colorectal tumorigenesis, we engineered a STAT3 Y705F knock-in to interrupt STAT3 activity in HCT116 and RKO colorectal cancer cells. These STAT3 Y705F mutant cells fail to respond to cytokine stimulation and grow slower than parental cells. These mutant cells are also greatly diminished in their abilities to form colonies in culture, to exhibit anchorage- independent growth in soft agar, and to grow as xenografts in nude mice. These observations strongly support the premise that STAT3 Y705 phosphorylation is crucial in colorectal tumorigenesis. Although it is generally believed that STAT3 functions as a transcription factor, recent studies indicate that transcription-independent functions of STAT3 also play an important role in tumorigenesis. We show here that wild-type STAT3, but not STAT3 Y705F mutant protein, associates with phospholipase Cg1 (PLCg1). PLCg1 is a central signal transducer of growth factor and cytokine signaling pathways that are involved in tumorigenesis. In STAT3 Y705F mutant colorectal cancer cells, PLCg1 activity is reduced. Moreover, overexpression of a constitutively active form of PLCg1 rescues the transformation defect of STAT3 Y705F mutant cells. In aggregate, our study identifies previously unknown cross- talk between STAT3 and the PLCg signaling pathways that may play a critical role in colorectal tumorigenesis. Mol Cancer Res; 9(10); 1418–28. Ó2011 AACR. Introduction tumorigenesis, because its mitochondrial activity appears to be required for Ras-mediated tumor transformation Signal transducer and activator of transcription 3 (5, 6). Third, transgenic mice with keratinocytes expressing (STAT3) is thought to be an oncogene (1). Several lines constitutively active STAT3 develop hyperproliferative of evidence support such a premise. First, persistent STAT3 dermatologic disorders in vivo (7). Fourth, targeted deletion activation has been detected in leukemia and in a variety of of STAT3 in skin cells prevents epithelial cancers in mice solid tumors including breast, brain, pancreas, ovarian, and (8), and targeting STAT3 specifically in B and T cells squamous cell carcinomas of head and neck (SCCHN) prevents development of lymphomas and myelomas (9). cancers, and melanomas (2). Second, constitutively active Latent cytoplasmic STAT3 becomes activated through STAT3 transforms rat and mouse cells and dominant phosphorylation of Y705 by cytoplasmic nonreceptor negative STAT3 blocks Src-induced transformation in vitro tyrosine kinases including Janus-activated kinase (JAK) (3, 4). Interestingly, recent studies show that the mitochon- and Src (10). Phosphorylated STAT3 dimerizes through drial functions of STAT3 may be important factors in reciprocal Src Homology 2 (SH2)-phosphotyrosine inter- action and accumulates in the nucleus (2). Therefore, STAT3 activates the transcription of a wide array of genes Authors' Affiliations: Departments of 1Genetics and Case Comprehen- 2 3 Bcl-XL sive Cancer Center, Epidemiology and Biostatistics, Medicine, School of including B-cell lymphoma-extra large ( ) and sup- Medicine, Case Western Reserve University; 4Genomic Medicine Institute, pressor of cytokine signaling 3 (SOCS3; ref. 2). Although Cleveland Clinic Foundation, Cleveland, Ohio; 5The Second Huaxi Hos- pital, Sichuan University, Chengdu; and 6College of Life Sciences, Wuhan the kinases that phosphorylate the Y705 residue of STAT3 University, Wuhan, China are well defined in epithelial and hematopoietic cells, the Note: Supplementary data for this article are available at Molecular Cancer phosphatases that specifically dephosphorylate pY705 have Research Online (http://mcr.aacrjournals.org/). received little attention. Corresponding Authors : Zhenghe Wang, Case Western Reserve Univer- We previously identified STAT3 as a direct substrate of sity, 10900 Euclid Avenue, WRB 3120, Cleveland, OH 44106-7285. Phone: protein tyrosine phosphatase receptor T (PTPRT; ref. 11). 216-368-0446; Fax: 216-368-8919; E-mail: [email protected] or Xiaodong Zhang, College of Life Sciences, Wuhan University, Luojia Hill, PTPRT is mutated in colon, lung, stomach, and skin Wuhan, P. R. China 430072. Phone/Fax: 86-27-68756606; E-mail: (melanoma) cancers (12). Moreover, PTPRT knockout mice [email protected] are highly susceptible to azoxymethane-induced colon doi: 10.1158/1541-7786.MCR-11-0147 tumors (13), indicating that PTPRT normally functions Ó2011 American Association for Cancer Research. as a tumor suppressor. Our finding that PTPRT specifically 1418 Mol Cancer Res; 9(10) October 2011 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst August 12, 2011; DOI: 10.1158/1541-7786.MCR-11-0147 Cross-talk between STAT3 and PLCg1 dephosphorylates STAT3 at the Y705 residue supports a days. The geneticin resistant clones were then screened for critical role for regulation of STAT3 Y705 phosphorylation homologous recombination by 35 cycles of genomic PCR in colorectal tumorigenesis. Although STAT3 is implicated with primers derived from the neomycin resistance gene in oncogenesis of leukemia, skin, and head and neck cancers 50-GTTGTGCCCAGTCATAGCCG-30 and the up- (1), the impact of STAT3 Y705 phosphorylation in colo- stream region of the left homologous arm 50-TCA- rectal tumorigenesis has not heretofore been well defined. GTTTCTTGGCCCAAAGT-30. Confirmatory genomic Here we show that successful knock-in (KI) of the STAT3 PCR was also carried out with positive clones identified by Y705F mutant allele into 2 different colorectal cancer cell primers derived from the neomycin resistant gene (50- lines results in mutant colorectal cancer cells that are less TCTGGATTCATCGACTGTGG-30) and the down- tumorigenic both in vitro and in vivo. The results of this stream region of the right homologous arm (50-TA- study further suggest that modulation of tumorigenicity is at GGCGCCTCAGTCGTATCT-30). The DNA fragments least partially dependent on STAT3 cross-talk with phos- from the confirmatory PCRs were then sequenced to pholipase Cg1 (PLCg1) through effects on S1248 phos- ensure the presence of the mutant Y705F alleles. To phorylation. PLCg1 is a key signaling molecule that target the second allele with the same targeting virus, hydrolyzes phosphatidylinositol-4,5-biophosphate to gener- correctly targeted clones were infected with adenoviruses ate inositol-1,4,5-triophosphate (IP3) and 1,2-diacylglycerol expressing the Cre-recombinase to delete the drug selec- þ (DAG), which, in turn, activate intracellular Ca2 and tion marker. To select clones with successful deletion of protein kinase C (PKC) signaling pathways that are impli- the drug selection marker, 30 cycles of genomic PCR were cated in tumorigenesis (14). In support, we show that carried out to amplify an approximately 200-bp genomic colorectal cancer cells carrying STAT3 mutated inY705 also fragment in which the Lox P site was inserted (using exhibit reduced PKC activities. primers 50-GCAGATGGAGCTTTCCAGAC-30 and 50- CGCCTGGGAAGAAGAAAAC-30). The heterozygous Materials and Methods KI clones were infected with the same targeting virus to target the second allele and the neomycin resistance gene Cell culture was excised as described earlier. HCT116, RKO, and HEK 293T cells were obtained from the American Type Culture Collection. HCT116 and Plasmid transfection RKO colorectal cancer cells were maintained in McCoy 5A Cells were plated 1 day prior to transfection to achieve a media plus 10% FBS. HEK 293T cells were maintained in 70% confluence at the time of transfection. Plasmids were Dulbecco's modified Eagle's medium media plus 10% FBS. transfected with the Lipofectamine Transfection Reagent (Invitrogen; catalogue no. 18324-020) according to the Somatic cell gene targeting manufacturer's instructions. To transfect a T75 flask of Somatic cell gene targeting was conducted as described HEK 293 cells, 9 mg of plasmids were mixed with 54 mLof (15, 16). Briefly, a 1.3-kb fragment from intron 21 to intron Lipofectamine Transfection Reagent and 1.5 mL of Opti- 22 of the STAT3 locus containing the exon 22 sequences MEM (Invitrogen; catalogue no. 31985070). The trans- was amplified 25 cycles from genomic DNA, using primers fection mixture was then incubated with cells at 37C for 4 50-TGACCAACTAGTCTGCTTACTGAATGCGAAG- hours. Cells were subsequently cultured in normal medium TCACAG-30 and 50-TGACCACCGCGGAGGGTCCT- after washing once with Hank's balanced buffer. TTCTCATTCCCACCTTA-30. The coding sequences for Y705 were then mutated from TAC (Tyr) to TTC Western blot (Phe) by site-directed mutagenesis, using primers 50-TC- Cells were lysed in radioimmunoprecipitation assay CCAGGCGCTGCCCCATTCCTGAAGACCAAGTT- (RIPA) buffer with complete protease inhibitor mixture TATC-30 and 50-AATGGGGCAGCGCCTGGGA-30. and phosphatase inhibitors [50 mmol/L Tris-HCl (pH 8.0), This mutated fragment was used as the right homologous 0.5% triton