Zuoxiang Xiao

The pivotal role of IKKα in the development of spontaneous lung squamous cell carcinomas.

Here, we report that kinase-dead IKKα knockin mice develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation. IKKα reduction upregulated the expression of p63, Trim29, and keratin 5 (K5), which serve as diagnostic markers for human lung SCCs. IKKα(low)K5(+)p63(hi) cell expansion and SCC formation were accompanied by inflammation-associated deregulation of oncogenes, tumor suppressors, and stem cell regulators. Reintroducing transgenic K5.IKKα, depleting macrophages, and reconstituting irradiated mutant animals with wild-type bone marrow (BM) prevented SCC development, suggesting that BM-derived IKKα mutant macrophages promote the transition of IKKα(low)K5(+)p63(hi) cells to tumor cells. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs.

Abstract 603: TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We recently reported that kinase-dead IKKα knockin mice, in which an ATP binding site of IKKα, Lys 44 was replaced by alanine (IkkαKA/KA), develop spontaneous lung SCCs that were closely associated with lung inflammatory microenvironment. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs (Xiao et al. Cancer Cell 23(4):527-40, 2013). To further elucidate what and how the inflammatory stimuli contribute to the tumorigenesis, we generated bitransgenic mice harboring IkkαKA/KA and tnfrsf1-/- (KALTFR). It was identified that the KALTFR mice display lung tumor-free even the lung inflammations are still severe, while almost all of the littermate IkkαKA/KA mice developed lung SCCs. Interestingly, we found a noticeably increased TNFR1 expression in the lung SCCs of IkkαKA/KA mice compared to normal lung tissues. It has also been detected that the TNFR1 expressions are significantly higher in both transcript and protein levels in human lung SCCs with the compare to their adjacent tissues. These findings indicate that TNFR1 may play a crucial role in the development of lung SCC in either IkkαKA/KA mice or humans. Further investigation demonstrated that TNFR1 is a critical factor to regulate nuclear transcriptional factors and oncogenes including C-Myc, through one bromodomain (BRD) proteins- containing complex. These findings broaden our understanding about how inflammatory microenvironment contributes to tumorigenesis through TNFα-TNFRs engagements. Citation Format: Zuoxiang Xiao, Jami Willette-Brown, Yinling Hu, Sichuan Xi. TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas. [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 603. doi:10.1158/1538-7445.AM2014-603

Abstract 3618: Ikkα modulates primary sclerosing cholangitis and intrahepatic cholangiocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer after hepatocellular carcinoma (HCC). The risk of ICC is higher in patients with primary sclerosing cholangitis (PSC). To date, the cellular and molecular mechanism underlying the pathological progression of PSC and ICC is poorly understood. IKKα is part of the IκB kinase (IKK) complex, which plays an important role in regulating inflammation- associated carcinogenesis through both NF-κB-dependent and independent pathways. Here, we show that IKKα mutant mice developed very serious PSC as early as four weeks of age. The ALT/AST and bilirubin levels were significantly increased in the serum of IKKα mutant mice along with lymphocytic and eosinophilic infiltration into the liver. Liver inflammation in the IKKα mutant mice, mediated by macrophages, neutrophils and CD4+ T cells, was associated with the death of cholangiocytes and hepatocytes, and obstruction of intrahepatic and extrahepatic bile ducts, which impeded bile flow and ultimately led to biliary fibrosis and cirrhosis. Additionally, upon activation of NOTCH signaling in the liver via hydrodynamic shear, we observed that NOTCH-induced ICC, with the PSC, developed significantly faster in IKKα mutant mice. To identify whether intrinsic IKKα dysfunction in hepatocytes promotes the NOTCH-induced ICC in IKKα mutant mice, we generated IKKα hep KO mice, in which IKKα is conditionally knocked out in hepatocytes. No biliary disease or liver injury was observed in these mice. We then established an accelerated ICC model utilizing hydrodynamic delivery of NICD and AKT expression vectors. Unexpectedly, ICC development was remarkably slower in the IKKα hep KO mice compared to the IKKα floxed control mice, and this delayed ICC development was associated with reduced activation or levels of AKT, NOTCH, MAPK/ERK and c-Myc. These data suggest that IKKα may play a protective role in PSC, while promote ICC derived from hepatocytes. In contrast to ICC, we also established the cMET/β-catenin-induced HCC model in the IKKα hep KO mice, which did not reveal any difference in tumor development between IKKα hep KO and control mice. Taken together, our findings suggest that IKKα plays complicated and important roles in PSC and ICC pathological progression. Citation Format: Qun Jiang, Zuoxiang Xiao, Timothy Back, Anthony Scarzello, Scott Roan, Jami Willette-Brown, Feng Zhu, Yinling Hu, Robert H. Wiltrout. Ikkα modulates primary sclerosing cholangitis and intrahepatic cholangiocarcinoma. [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 3618. doi:10.1158/1538-7445.AM2014-3618

Abstract 1083: The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas.

During the last decades lung cancer has become the leading cause of cancer deaths in the world, and the need to develop better diagnostic techniques and therapies is urgent. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, there are no robust animal models of lung squamous cell carcinomas (SCCs), one of the major types of lung cancer. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. All the knock-in mice at 4 to 6 months of age developed spontaneous lethal lung SCCs associated with markedly increased leukocyte infiltration and expression of cytokines, and chemokines in the lungs. Furthermore, we identified deregulated c-myc, Nanog, Oct3/4, p53, Rb, EGFR, MAPK, Jun-B, p63, Trim29, Rhov, CDK1, and IGF1 in mouse lung SCCs, and identified reduced IKKα and IκBα and increased ROS1 in mutant lungs and SCCs, some of which were found in human lung SCCs. Lung cancers were prevented by reintroducing epithelial-cell IKKα, depleting macrophages or depleting lymphocytes. This study not only provides a novel model for studying the pathogenesis, treatment, early detection, and prevention of human lung SCCs, but also demonstrates how a single mutation in IKKα elicits malignancy through the combined epithelial-cell-autonomous and immune mechanisms. Citation Format: Zuoxiang Xiao, Qun Jiang, Jami Willette-Brown, Feng Zhu, Sichuan Xi, Sandra Burkett, Fanching Lin, Timothy Back, Mahesh Datla, Zhonghe Sun, Romina Goldszmid, Xiaolin Wu, David Schrump, Howard Young, Georgio Trinchieri, Robert Wiltrout, Yinling Hu. The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2013-1083

Abstract 3071: Epigenetic repression of miR-217 contributes to tobacco-induced esophageal carcinogenesis.

Although alterations in microRNA (miRNA; miR) expression have been implicated in the pathogenesis of a variety of human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In order to examine this issue, array and RT-PCR techniques were used to examine miRNA expression profiles in immortalized esophageal epithelial cells (Het-1A), as well as NCI-SB-EsC1 (EsC1), NCI-SB-EsC2 (EsC-2), OE19, and OE33 esophageal cancer cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression (1.5-9.2 fold) in esophageal cancer cells and Het-1A cells. Endogenous miR-217 expression levels in cultured esophageal cancer cells were significantly lower than those observed in Het-1A cells. Consistent with these findings, miR-217 was significantly down-regulated (3.5-23.5 fold) in resected esophageal cancers relative to adjacent normal esophageal tissues. Software-guided analysis revealed that miR-217 potentially targeted KLK7, encoding a kallikrein family member implicated in invasion and metastasis of several cancers. Constitutive over-expression of miR-217 inhibited expression, whereas depletion of endogenous miR-217 enhanced expression of KLK7 in Het-1A, EsC1 and EsC2 cells. RNA cross-link immunoprecipitation (CLIP) experiments confirmed direct interaction of miR-217 with KLK7 transcripts. Methylated DNA Immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP) experiments demonstrated that CSC increased DNA methylation and decreased H3K4me3 levels within the miR-217 genomic locus in Het-1A, EsC1 and EsC2 cells. Deoxyazacytidine (DAC) induced miR-217 expression in EsC1 and EsC2 cells but not Het-1A cells, and markedly attenuated CSC-mediated miR-217 repression in these cells. Over-expression of miR-217 significantly decreased proliferation of esophageal cancer and Het-1A cells, as well as invasion of esophageal cancer cells. Depletion of miR-217 increased growth of Het-1A cells, but not EsC1 and EsC2 cells presumably due to lower endogenous miR-217 expression in these cancer cells. Experiments are currently in progress to examine the effects of miR-217 and KLK7 expression on tumorigenicity of esophageal cancer cells. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of esophageal carcinomas, and suggest that restoration of miR-217 expression may be a novel strategy for therapy of these malignancies. Citation Format: Sichuan Xi, Suzanne Inchauste, Zuoxiang Xiao, Jigui Shan, Mary Zhang, Julie A. Hong, Manish T Raiji, David G. Beer, David S. Schrump. Epigenetic repression of miR-217 contributes to tobacco-induced esophageal carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2013-3071 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2560: IKKα inactivation predisposes to spontaneous lung squamous cell carcinomas

Lung cancer is the leading cause of cancer deaths worldwide. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, most animal models resemble human lung adenocarcinoma, and spontaneous lung squamous cell carcinomas (SCCs) mouse models are very rare. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. The knock-in mice develop severe skin lesions and begin to die after 6 months. We found lung SCCs in some of the mice. To study lung SCC development, we decided to stabilize the skin condition by reintroducing transgenic IKKα by crossing KA/KA with Lori.IKKα transgenic mice to generate KA/KA/Lori.IKKα (KA/KA L ) mice. Almost all the KA/KA L (100%) mice at 4 to 6 months of age developed spontaneous lethal lung SCCs. The endogenous IKKα protein level generally markedly declined in an age dependent manner in these IKKα mutant mice. Progenitor cell related markers Sox2, OCT3/4 and Nanog were only increased in the lung SCC tissue but not in the tumor adjacent tissues, implying the involvement of cancer stem cells in lung SCC. Furthermore, we detected substantial increases in Ras and CyclinD1 levels and EGFR, ERK and p38 activities in lung SCCs. On the other hand, we detected reduction in tumor suppressor gene Rb and IαBβ accompanying with reduced IKKα levels in KA/KA L lungs as well as in lung SCCs. Importantly, we observed a similar alteration pattern in mouse and human lung SCCs. Finally, reintroducing IKKα into lung epithelial cells prevented lung SCC development in mice. Collectively, our study supports the tumor suppressing role of IKKα in lung tumorigenesis. This novel lung SCC mouse model may facilitate investigations in pathogenesis, diagnosis, and treatment of human lung SCC disease. 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 2560. doi:1538-7445.AM2012-2560