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Colorectal cancer (CRC) is one of the most common causes of cancer deaths in the world, and most of CRC arise sporadically by the emergence of multiple chromosomal aberrations. Allelic losses in the long arm of chromosome 4 are commonly encountered in many human malignancies, but few tumor suppressor genes (TSGs) are identified. By loss of heterozygosity study at chromosome 4 in 114 colorectal carcinomas, we identified N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 4 (NDST4) as a novel TSG that had been published recently. We demonstrated for the first time that the genetic loss of NDST4 was significantly associated with advanced pathological stage and poorer overall survival of patients. Meanwhile, gene expression of NDST4 was obviously decreased in 30 (57.7%) of 52 CRC tumors when compared with their matched normal mucosae. In the present study, tumor suppressor activity of NDST4 was identified by in vitro cell models and mouse xenograft tumor models. Ectopic expression of NDST4 in the human CRC cells induced a significant suppression in cell proliferation, anchorage-independent growth and invasion in vitro. Subcutaneous injection of NDST4-inducible CRC cells in nude mice showed alleviated tumor growth in doxycycline treated mice when compared with doxycycline untreated mice. More importantly, via intrasplenic implantation, re-expression of NDST4 in silenced cells restrained liver metastasis, and also improved survival in nude mice. Taken together, these results indicate that NDST4 is a tumor suppressor gene associated with CRC, and its downregulation might promote tumor progression and distant metastasis. In addition, allelic loss of NDST4 gene could serve as a molecular predictor of metastasis and poor prognosis in patients with CRC. Citation Format: Tzu-Ming Jao, Ming-Hong Tsai, Sheng-Tai Tzeng, Ya-Chien Yang. N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 4, a novel tumor suppressor, suppresses tumorigenesis and liver metastasis of colorectal cancer cells in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2015-2149

NDST4 is one member of N-deacetylase/N-sulfotransferase (heparan glucosaminyl) (NDST) family, which are responsible for heparan sulfate (HS) biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs). The HS chains of HSPGs bind to and regulate their functions of various growth factors, cytokines and chemokines. HSPGs ubiquitously reside on cell surface, inside the cell, and in the extracellular matrix. Importantly, the content and distribution of HSPGs are altered during tumorigenesis. In the study, 30 (57.7%) of 52 primary colorectal carcinomas showed a dramatic reduction in NDST4 RNA transcripts, and genetic loss of NDST4 was significantly associated with poor survival of patients with colorectal cancer. We further generated an Ndst4-knockout mouse strain, which could develop and reproduce normally. Notably, using the dextran sodium sulfate mouse model of acute colitis, Ndst4 deficiency resulted in a significantly higher disease activity index and obviously hyperemic appearance in the cecum. In the development of colitis-associated cancer, the size of colonic tumors induced by azoxymethane/dextran sodium sulfate was significantly increased in Ndst4-deficient mice compared with wild-type mice. Taken together, loss-of-function of NDST4 might impair the modification of HS chains of specific HSPGs leading to tumor-promoting inflammation in the colon. The full spectrum of signaling pathways contributed by NDST4 to colitis and tumor progression remains to be elucidated. Citation Format: Tzu-Ming Jao, Ming-Hong Tsai, Chi-Yen Huang, Sheng-Tai Tzeng, Jing-Xing Lee, Wei-Chen Lo, Che-Wei Chang, Ya-Chien Yang. Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2014-2472

Shisa3, a newly identified tumor suppressor, inhibits invasion and metastasis via regulation of epithelial-mesenchymal transition in non-small cell lung cancer. We first screened 11 colorectal cancer (CRC) cell lines as well as 10 pairs of primary tumors and matched normal mucosas for Shisa3 expression by semi-quantitative RT-PCR. Shisa3 gene was silenced or down-regulated in 8 (72.7%) cell lines and 8 (80%) CRC tumors, suggesting that Shisa3 might be also involved in colorectal tumorigenesis. Accordingly, we further confirmed Shisa3 expression was significantly decreased in 59.6% (31/52) of colorectal carcinomas compared with corresponding normal mucosas by real-time RT-PCR (P < 0.001). Using gene copy number assay, we ruled out chromosomal instability pathway as the major mechanism resulting in Shisa3 silencing in CRC. While Shisa3 mRNA expression was restored in 66.7% (4/6) of CRC cell lines as exposure to DNA methylation inhibitor 5-Aza-2′-deoxycytidine. CpG islands spanning around Shisa3 gene were predicted via MethPrimer program. Aberrant methylation of 5 CpG loci in Shisa3 intron 1 was identified in CRC cell lines and primary tumors by bisulfite sequencing, and their methylation levels determined by pyrosequencing were inverse correlation with Shisa3 mRNA expression. In addition, among 127 pairs of CRC tissues, Shisa3 methylation levels in tumors were much higher than that in corresponding normal mucosas (P < 0.001), and Shisa3 hypermethylation was significantly associated with disease recurrence in stages B and C patients (P = 0.008). Kaplan-Meier survival analysis showed that patients with Shisa3 hypermethylation revealed significantly shortened overall survival (P < 0.001) and disease-free survival (P = 0.006). In conclusion, Shisa3 gene hypermethylation could serve as a molecular predictor of disease recurrence and poor prognosis in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4024. doi:1538-7445.AM2012-4024

Colorectal cancer (CRC) is one of the most common malignancies in the world. Many molecular abnormalities have been reported in CRC, of which accumulated alterations of tumor suppressor genes (TSGs) and proto-oncogenes are required for the disease development. Genomic deletion is reflected in a genetic mechanism called loss of heterozygosity (LOH). Allelic deletion at tumor suppressor loci is common in all human cancers, and contributes to TSG silencing. By allelotyping with 22 polymorphic microsatellite markers spanning from chromosome 4pter to 4qter, we first defined the most frequent LOH region at 4q27, which occurred allelic imbalance in 32.1% (34/106) of colorectal carcinomas. After fine deletion mapping in these carcinomas, we further identified a minimal region harboring one gene with known functions, named NDST4. By quantitative RT-PCR, NDST4 expression could hardly be detected in 10 of 11 CRC cell lines, and was downregulated in 57.7% (30/52) of CRC tumors. Importantly, NDST4 expression was significantly decreased in tumors compared with matched normal mucosa or adenomas (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4598. doi:1538-7445.AM2012-4598

Colorectal cancer (CRC) is one of the leading causes of cancer death in the world. By loss of heterozygosity approach, we identified Protocadherin 10 (PCDH10) gene with a high frequency of allelic deletion in CRC. Furthermore, the genetic alteration was associated with presence of distant metastasis and poorer overall survival of patients. Importantly, the expression of PCDH10 mRNA was silenced or downregulated in 37 (84.1%) out of 44 colorectal tumors as compared with their paired normal mucosa. We proposed that PCDH10 might be a CRC-associated tumor suppressor gene, and then investigated the biological functions of PCDH10 in CRC cells. First of all, re-expression of PCDH10 in HCT116 cells reduced cell proliferation, migration, invasion and anchorage-independent growth in vitro. Meanwhile, stable PCDH10- expressing cells (HCT116/PCDH10) exhibited suppression of tumorigenicity and liver metastasis in xenograft tumor models. In SCID mice, HCT116/PCDH10 cells with subcutaneous injection showed reduced tumor growth, and, with intrasplenic injection, showed reduced metastatic incidence and nodules in the liver, as compared with mock control cells. Taken together, these results indicate that PCDH10 is a tumor suppressor gene associated with CRC, and its downregulation might promote tumor progression and distant metastasis. In addition, allelic loss of PCDH10 gene could serve as a molecular predictor of metastasis and poor prognosis in patients with CRC. 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 2187. doi:10.1158/1538-7445.AM2011-2187