Zhijie Xiao

Abstract 4787: The role of CaMK2A in reactive oxygen species homeostasis in lung adenocarcinoma

The role of Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) in reactive oxygen species homeostasis in lung adenocarcinoma Lung cancer is the most common cancer worldwide and patients have poor prognosis and low survival rate. The unsatisfactory outcomes of lung cancer treatment are related to multiple factors including tumor recrudescence, metastasis, cancer drug resistance, etc. Cancer stem cells are cell subsets within a cancer that excel in tumor properties, especially survival, renewal and resistance to treatment. Reactive oxygen species (ROS) are high energy molecules produced during cellular hyperproliferation and cell death. Studies have shown though a low level of ROS is required to support stem cell functions but excessive ROS damages DNA and predisposes to cell death. Therefore, the ability to attenuate oxidative damage from ROS and maintain redox homeostasis is an important property of aggressive and resilient cancer cells. The multifunctional serine/threonine kinase calcium/calmodulin- dependent protein kinase II alpha (CaMK2A) is a calcium signaling molecule involved in cell growth and stress signals integration. It has been reported to play a tumor-supportive role in osteosarcoma, prostate cancer and leukemia. However, little is known about the impact and mechanisms of CaMK2A on lung cancer and cancer redox homeostasis. This study aims to investigate the role of CaMK2A in ROS regulation in lung cancer stem cells. We measured ROS levels using H2DCFDA probes in cancer stem cells isolated from two lung cancer cell lines by the known lung cancer stem cell marker CD44 and CD166. Compared to CD44- and CD166- non-stem cells, ROS levels were lower in both CD44+ and CD166+ cancer stem cells. In cancer cells with stable CaMK2A knockdown by shRNA (CaMK2A-KD), ROS was suppressed while CaMK2A overexpression led to ROS elevation, suggesting a potential role of CaMK2A on ROS regulation in lung cancer stem cells. To understand the mechanism, activated NRF2 levels, the master transcription factor mediating ROS regulation, were measured in total nuclear protein extracts. By Western blot, nuclear NRF2 protein showed upregulation in CaMK2A overexpression cells and decreased levels in CaMK2A-KD cells, implying NRF2 might be a mediator of CaMK2A in ROS regulation. To further identify downstream regulators, mRNA expression analysis of five ROS-regulatory NRF2 target genes (GSTP1, NQO1, GCLC, HMOX1, TRXR1) was performed. Quantitative PCR results showed significant corresponding changes of some these gene expressions in cell lines, which upregulated in CaMK2A overexpression and downregulated in both CaMK2A-KD cells and CaMK2A pharmacological inhibition cells. Together, the results indicate CaMK2A might regulate ROS status through mediating downstream target genes of NRF2. Citation Format: Siqi Wang, Zhijie Xiao, Vicky Tin, Ian K. Lam, Xuyuan Gao, Maria Wong. The role of CaMK2A in reactive oxygen species homeostasis in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2017-4787

Abstract LB-274: Whole-exome sequencing reveals tyrosine kinase-resistant mutations in pretreatment EGFR-mutant lung adenocarcinomas

Lung cancer is the most lethal malignancy around the world. In Hong Kong, chronic tobacco use is prevalent in male but Note: This abstract was not presented at the meeting. Citation Format: Xu-yuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P. Wong. Whole-exome sequencing reveals tyrosine kinase-resistant mutations in pretreatment EGFR-mutant lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-274. doi:10.1158/1538-7445.AM2017-LB-274

Abstract 2482: The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation

Tumor initiating cells (TIC) are a small population of cancer cells with enhanced tumorigenicity and drug resistance. Their stemness properties could be activated by stress-induced cell plasticity but the mechanisms remain to be studied. Calcium signaling is important for integrating cell responses to intra- and extracellular stresses. The role of its downstream pathway, calcineurin/NFAT, in lung TIC has not been explored. This study investigated the role and mechanisms of calcineurin/NFAT in regulating TIC and drug resistance by analyzing NFATc2 expression in multiple TIC populations and functional evaluation using in vitro and in vivo models. In tumor spheres and TIC isolated by marker-based flow cytometry, NFATc2 showed overexpression and higher luciferase reporter activities. Knockdown of NFATc2 reduced TIC marker proportions and suppressed TIC properties including pluripotency genes expression, sphere formation, cell mobility and drug resistance in vitro. Xenograft models and limiting dilution assay showed shNFATc2 effectively suppressed tumorigenesis and reduced TIC frequency. Stable NFATc2 knockdown sensitized tumors to cisplatin and gefitinib treatment. In contrast, TIC-supportive effects were observed in vitro and in vivo when NFATc2 was overexpressed. Further, NFATc2 was activated in stable gefitinib or cisplatin resistant cell lines, and knockdown of NFATc2 sensitized them to drug treatment. These data implicated NFATc2 is involved in lung TIC maintenance and drug resistance. To further investigate the molecular mechanisms of how NFATc2 mediate these roles, we analyzed pluripotency genes and observed a significant correlation between NFATc2 and SOX2 expression in lung cancer cell lines and human lung carcinomas. Computational analysis predicted NFATc2 binding sequences in SOX2 regulatory regions. Detailed studies including luciferase reporter assay, site-directed mutagenesis and CHIP-qRCR confirmed NFATc2 could regulate SOX2 expression through binding to its enhancer. Furthermore, immunohistochemistry on human lung cancers showed significant correlation between expressions of SOX2 and the anti-oxidative stem cell marker ALDH, suggesting SOX2 regulates ALDH expression. In vitro, NFATc2 knockdown suppressed ALDH1A1 expression but it was rescued by SOX2 overexpression. Vice versa, ALDH1A1 was upregulated in NFATc2 overexpressing cells but this was prevented by SOX2 knockdown. Functionally, reactive oxidative species (ROS) levels assessed by flow cytometry were suppressed by activated NFATc2/SOX2/ALDH1A1 axis, while treatment with the ROS inhibitor N-acetylcysteine reversed the sensitization of shNFATc2 on cisplatin treatment. Together, the data showed NFATc2 is involved in the regulation of TIC phenotypes in lung cancer. It increases drug resistance by ROS scavenging mediated by the SOX2/ALDH1A1 axis. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky PC Tin, Maria P. Wong. The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2482.

Abstract 3352: Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma

Lung cancer is the most lethal of all cancers world-wide and the majority of patients require chemotherapy or targeted therapy at presentation. Survival is compromised by drug resistance through multiple mechanisms such as by-pass mutations, micro-environment activation of survival programs and induction of tumor initiating cells (TIC). Induction of TIC phenotypes could be accompanied by upregulation of embryonic or developmental pathways but molecular mechanisms are incompletely understood. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) is a multifunctional serine/threonine kinase involved in growth and stress signals integration. A tumor-supportive role has been reported in leukaemia and thyroid cancer but reports on its involvement in TIC regulation are limited. We investigated the role and molecular mechanism of CaMK2A in lung cancer TIC regulation using in vitro and in vivo models. The results showed CaMK2A was overexpressed in lung cancer cell lines and human lung adenocarcinomas compared to normal lung. In cancer cells with stable CaMK2A-knockdown, in vitro reduction in tumor spheres formation, anchorage independent growth and increased reactive oxygen species, as well as in vivo reduction of xenograft tumorigenicity were observed. The opposite effects were observed in cancer cells with stable CaMK2A over-expression. Further, specific pharmacological inhibition of CaMK2A by KN93 led to significantly reduced IC50 for gefitinib in lung cancer cells harboring activating EGFR mutation (HCC827) and in HCC827 exogenously induced for gefitinib resistance. In contrast, IC50 of cisplatin treatment was increased in cancer cells with genetically-induced CaMK2A over-expression. Moreover, CaMK2A knockdown led to reduced mRNA expressions of pluripotency genes (SOX2, NANOG, OCT4) and TIC markers (ALDH, CD166). These results suggested CaMK2A is involved in lung tumorigenicity through TIC regulation. To further investigate the molecular mechanism, we showed tumorigenicity was restored in xenografts with CaMK2A knockdown rescued with SOX2 over-expression. In CaMK2A knockdown cells, reduced SOX2 expression was associated with increased H3K27me3 repressive histone mark, which has not been reported in the literature. Taken together, we have shown CaMK2A plays a role in lung adenocarcinoma and TIC maintenance through histone modification and regulation of SOX2 expression. Targeting TIC through CaMK2A modulation might be useful for overcoming drug resistance and improving long term lung cancer survival. Citation Format: Maria P. Wong, Jing Liu, Zhi-Jie Xiao, Si-Qi Wang, Vicky Pc Tin. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3352.

Abstract 1949: P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma

The p38 MAPK cascade regulates body responses to a wide range of stress stimuli in a context-dependent and tissue-specific manner. The isoform p38alpha (p38) mediates both tumor suppressive and tumor-supportive functions in different cancer models. However, its role in lung cancer is not clear. Since EGFR is frequently mutated in lung adenocarcinomas in non-smoking women and p38 is a downstream target of EGFR signaling, it is possible that p38 plays a tumor-supportive role. Using immunohistochemistry to analyze nuclear activated p38 expression in 90 lung adenocarcinomas, we observed p38 expression was significantly associated with well differentiated compared to poorly-differentiated carcinomas (p Citation Format: Jing Liu, Lai-han Leung, Zhi-jie Xiao, Vicky PC Tin, Maria Pik Wong. P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma. [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 1949. doi:10.1158/1538-7445.AM2014-1949

Abstract LB-271: The CD44high/ALDHhighcell population mediates tumor initiating cell properties through the sonic Hedgehog pathway in lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In spite of the decreasing lung cancer incidence recently observed in some countries, its mortality has remained high due to late presentation and limited chemotherapy effectiveness. Even for EGFR mutant cancers initially responding to tyrosine kinase inhibitors (TKI), long term patient survival has not been achieved due to the almost universal occurrence of TKI resistance. Tumor initiating cells (TIC) are a subpopulation of cancer cells believed to be capable of self-renewal leading to tumor perpetuation, metastasis and treatment resistance. To identify self-renewing cells in lung cancer and investigate their regulatory pathways, tumor cells with differential expression of the combined CD44 and ALDH1 markers were isolated by flow cytometry for functional comparison. The results showed in established lung cancer cell lines and early passage clinical lung cancer cells, the CD44high/ALDHhigh population had the strongest, while CD44low/ALDHlow the weakest in-vitro cell sphere formation and in-vivo serial xenograft transplantation capacities. The CD44high/ALDHhigh population also showed higher matrigel invasion ability, pluripotency genes expression, cell cycle regulator expression, as well as resistance to tyrosine kinase inhibitor and/or cisplatin treatment. TKI treatment led to increased CD44high/ALDHhigh cell proportions. Simultaneous down-regulation of CD44 by si-RNA and enzymatic inhibition of ALDH1 suppressed stem/progenitor genes expression including OCT4, SOX2 and NANOG, and attenuated cell sensitivity to TKI and cisplatin treatment in different lung cancer cells, indicating CD44 and ALDH were not only surface markers for TIC, but also had functional roles in TIC maintenance. To investigate for phenotypic differences and potential regulatory pathways involved in the maintenance of the two cell populations, the expression microarray profiles of isolated TIC (CD44high/ALDHhigh) and non-TIC (CD44low/ALDHlow) populations were compared. The results showed differential expression of various cell adhesion molecules, HLA markers, and signaling proteins involved in the Sonic Hedgehog (e.g. GLI1, GLI3) and Notch (e.g. DLL4, JAG1) pathways. Differential expression of selective molecules was independently validated by quantitative RT-PCR in an expanded panel of lung cancer cell lines. Pharmacological inhibition of the Hedgehog pathway using cyclopamine decreased TIC proportion in a dose dependent manner and sensitized lung cancer cells to TKI treatment, while Notch inhibition led to less prominent effects. Taken together, these findings indicated that CD44high/ALDHhigh expression characterized a subpopulation of self-renewing and tumorigenic cells in lung cancer. Therapeutic targeting of the Hedgehog pathway might be useful for disrupting TIC and help to achieve lung cancer control. Citation Format: Maria Pik Wong, Jing Liu, Zhi-jie Xiao. The CD44high/ALDHhigh cell population mediates tumor initiating cell properties through the sonic Hedgehog pathway in lung cancer. [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 LB-271. doi:10.1158/1538-7445.AM2013-LB-271