Jeng-Fan Lo

Network Biology of Tumor Stem-like Cells Identified a Regulatory Role of CBX5 in Lung Cancer

Mounting evidence links cancers possessing stem-like properties with worse prognosis. Network biology with signal processing mechanics was explored here using expression profiles of a panel of tumor stem-like cells (TSLCs). The profiles were compared to their parental tumor cells (PTCs) and the human embryonic stem cells (hESCs), for the identification of gene chromobox homolog 5, CBX5, as a potential target for lung cancer. CBX5 was found to regulate the stem-like properties of lung TSLCs and was predictive of lung cancer prognosis. The investigation was facilitated by finding target genes based on modeling epistatic signaling mechanics via a predictive and scalable network-based survival model. Topologically-weighted measurements of CBX5 were synchronized with those of BIRC5, DNMT1, E2F1, ESR1, MLH1, MSH2, RB1, SMAD1 and TAF5. We validated our findings in another Taiwanese lung cancer cohort, as well as in knockdown experiments using sh-CBX5 RNAi both in vitro and in vivo.

Abstract 2293: Deletion of Tid1 in hepatocyte promotes steatosis, fibrosis, and tumorigenesis

Background: Tid1, DnaJ cochaperone protein, is a mammalian homolog of the Drosophila tumor suppressor Tid56 whose anti-tumor function is through its capacity to regulate cell differentiation in imaginal discs. In our previous studies, we found Tid1 regulates early mouse embryogenesis and T cell development. In addition, it has been identified that Tid1 can act as a tumor suppressor in breast, colon, lung and head and neck cancer. Tid1 protein is highly expressed in mouse and human hepatocyte. However, the physiological role of Tid1 in hepatocyte development and hepatic tumorigenesis remains elusive. Methods: Mice with loss of Tid1 lead to embryonic lethality as early as E4.5. Therefore; we generated a hepatocyte specific Tid1 knockout mice by crossing the Tid1flx/flx mice generated in our lab with transgenic mice expressing Cre recombinase activity driven by albumin promoter. Both spontaneous or carcinogen (diethylnitrosamine; DEN) mediated hepatocellular carcinoma (HCC) in Tid1 deficiency mice will be monitored. In addition, the physiological function of hepatocytes including albumin, plasma alanine aminotransferase (ALT), triglyceride (TG) and cholesterol collected from the control and mutant mice will be examined. Results: We have successfully established mice with hepatocyte specific ablation of Tid1 mice. Immunohistochemistry staining and western blot showed that conditional knockout mice were deficient on Tid1 expression in hepatocyte. Deletion of Tid1 in hepatocyte caused the hepatic steatosis and fibrosis. Consequently, spontaneous HCC tumor was found in thirteen-month-old Tid1 knockout mice. In DEN-treated mice model, deletion of Tid1 also enhanced the induction of HCC accompanying with elevatiof of plasma ALT and cholesterol. Conclusion: The progression of steatosis, fibrosis to HCC was observed in Tid1 deficient mice, which similarly reflects to those observed in tumorigenesis of human HCC. Therefore, we could further characterize the molecular mechanisms to understand how human HCC is developed through our Tid1-deficent HCC mice model. Ultimately, therapeutic regime for HCC could be developed from the Tid1-deficent mice for improving future HCC therapy. Citation Format: Yu-Syuan Chen, Jeng-Fan Lo. Deletion of Tid1 in hepatocyte promotes steatosis, fibrosis, and tumorigenesis. [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 2293. doi:10.1158/1538-7445.AM2015-2293

Abstract 91: Characterization of Tid1 conditional knockout mice for preclinical testing of novel human head and neck cancer therapeutics

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBackground: Head and neck cancer (HNC) includes oral cancer (OC). Due to high recurrence, high mortality and resistance to conventional therapies, the development of new chemopreventive agents for HNSCC is an important research priority. Tid1, a DnaJ cochaperone protein, interacts with ErbB family members including EGFR and ErbB2, which function as oncogenes during the tumorigenesis of head and neck cancer and breast cancer. Down regulation of activity or expression level of EGFR and ErbB2 is conversely correlated to expression of Tid1 in head and neck cancer and breast cancer, respectively. In addition, downregulation of Tid1 causes the upregulation of interleukin- 8 (IL-8) to enhance the metastatic ability in breast cancer cells. Transgenic mouse modeling has been an instrumental tool for gene specific physiological and functional study. Additionally, we have demonstrated that Tid1 is important for T cell development and early embryogenesis by established knockout mice systems.nnMethods: We plan to generate the transgenic mouse models illustrating the head and neck cancer carcinogenesis with our Tid1 knockout mice. Carcinogen administration, drinking water accompanying with 4-NQO (4-Nitroquinoline 1-oxide), has been incorporated within the transgenic mouse modeling along with our Tid1 deficiency mice, at which Tid1 deficiency will be a promoting “hit” during the progression of tumorigenesis with requirement of extra “hits”.nnResults: Currently, we have successfully established mice with epithelial specific ablation of Tid1 by crossing Tid1flx/flx mice with transgenic K5-Cre mice. Immunohistochemistry or immunefluorescent staining were applied to showed that mutant mice were deficient on Tid1 expression in epidermal layers. Further, the mutant and control mice were subject to carcinogen 4-NQO treatment within drinking water for modeling head and neck cancer progression. Up to date, we discovered that mutant mice treated with 4-NQO displayed faster incident of neoplasia in oral cavity and reduction of body weight in comparison to control mice under same administration. In addition, the histopathological analyses showed that the tumors formed in mutant mice were benign squamous papilloma.nnConclusion: These tumors generated in mutant mice exhibited changes in the expression pattern of keratins similar to those observed in human premalignant oral tumors, which are reflective of early stages of tumorigenesis. Therefore, we can further characterize the histopathology and molecular mechanisms of primary or metastatic head and neck cancer development from above mouse models. With the mouse models generated and comprehensiveness with the clinical relevance from our newly identified findings, we will conduct the long-term objectives for the development of therapeutic intervention, which will be based on the above findings to ensure the efficacy of testing drugs at the pre-clinical stage.nnCitation Format: Jeng-Fan Lo, Li-Hao Cheng. Characterization of Tid1 conditional knockout mice for preclinical testing of novel human head and neck cancer therapeutics. [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 91. doi:10.1158/1538-7445.AM2014-91

Abstract 1705: Combinatorial therapeutics with targeting head and neck cancer initiating cells using active components from antrodia cinnamomea and conventional chemotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBackground: Cancer initiating cells (CICs), also termed as Cancer stem cells (CSCs), represent a rare subpopulation of cells, which are responsible for tumor growth. CICs such as head and neck cancer initiating cells (HN-CICs), are often resistant to either radio- or chemotherapy. Therefore, development for drug candidates that target HN-CICs specifically will be effective for future oral cancer therapy.nnMethods: In this study, we used the in vitro cell-based aldehyde dehydrogenase (ALDH) activity assay system, which has been demonstrated as a CICs marker, to screen for the active purified components from Antrodia Camphorata Mycelia extract (ACME), which can target HN-CICs. Next, the antitumorigenicity of combinatorial treatment with active components from ACME and conventional chemotherapy on HNSCC will be examined in vitro and in vivo.nnResults: Herein, we first screened for the active components from natural products of ACM significantly down regulate the ALDH activity and CD44 positivity of Head and neck squamous cell carcinoma (HNSCC). Consequently, treatment of YMGKI-2 (analogue of ergosterol), a single and active compound purified from ACME, not only inhibited the ALDH activity but also reduced self-renewal property and expression of stemness genes (Oct-4 and Nanog) in enriched HN-CICs. Moreover, the repressive HN-CICs properties by YMGKI-2 treatment were caused by promoting cell differentiation. Additionally, treatment of YMGKI-2 results in down-regulation of Cox-2 and Src molecular mechanisms which both play important role in CICs. Further, the tumorigenic properties of HNSCC cells were attenuated by YMGKI-2 treatment in vivo. Lastly, combinatorial treatments with YMGKI-2 and standard chemotherapeutic drugs reveal synergistic effects on killing both HN-CICs and non-HN-CICs in vitro and in vivo.nnConclusion: Together, our results indicate that YMGKI-2 treatment significantly and selectively abolished the stemness properties and tumorigenicity of HN-CICs. These findings provide a new drug candidate from purified components of ACM as an alternative therapy in combination with conventional chemotherapy treatment modalities for head and neck cancer in the future.nnCitation Format: Ching-Wen Chang, Jeng-Fan Lo. Combinatorial therapeutics with targeting head and neck cancer initiating cells using active components from antrodia cinnamomea and conventional chemotherapy. [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 1705. doi:10.1158/1538-7445.AM2014-1705

Abstract 3338: Characterization of glucose transporter 3 in head and neck cancer initiating cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnBackground: Head and Neck squamous cell carcinoma (HNSCC) is a lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Cancer initiating cells (CICs) exhibit self-renewal and promote tumor progression capacity. We have also identified the subpopulation of head and neck cancer initiating cells (HN-CICs), and observed the upregulation of Glucose Transporter 3(Glut3) in HN-CICs by differential systemic analyses. However, the role of Glut3 in HN-CICs metabolism alternation remains unclear. Herein we determined the critical role of Glut3, a novel CICs marker, in the maintenance of stemness characteristics and tumorigenic phenotype of HN-CICs. Methods: Stable overexpression and knockdown of Glut3 expression in HNSCCs and HN-CICs was achieved by lentiviral-mediated system. Consequently, we elucidated the stemness properties and tumorigenicity of HNSCCs and HN-CICs with Glut3 down-regulation. Consequently, we investigated the role of Glut3 in metabolism regulation of cancer initiation cells. Results: Lentiviral knockdown of Glut3 significantly reduced the self-renewal ability and cancer initiation cell marker Grp78 expression in HN-CICs. Additionally, down-regulation of Glut3 enhanced the differentiation capability but inversely diminished “stemness” gene expression of HN-CICs. Of note, knockdown of Glut3 lessened tumorigenicity of HN-CICs both in vitro. Conclusion: We showed that Glut3 contributes to the maintenance of stemness and tumorigenicity of HN-CICs. In addition, the expression of Glut3 was involved in HN-CICs metabolism. Overall, silencing Glut3 might be a potential therapeutic target for HNSCC by eliminating CICs.nnCitation 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 3338. doi:1538-7445.AM2012-3338

Abstract 3992: Tid1, a tumor suppressor, regulating the cancer initiation properties of HNSCC

Background: Tid1, a DNAJ/Hsp40 family protein, functions as a tumor suppressor in different types of cancer including HNSCC and influences many signaling pathway in cancer cells. Expression of Tid1 protein is negatively associated with the malignancy of HNSCC cells and tumor tissues. In addition, it has been shown that Tid1 interacts with P53, which exerts negative regulation of Nanog, one of the transcriptional factors to maintain the cellular stemness properties. Epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose their polarity and later acquire a migratory mesenchymal phenotype, is one of the crucial processes that induce tumor invasion and metastasis. Further, gain of EMT could promote stem cells (SCs) properties. Accumulating data support the hierarchical model of cancer initiating cells (CICs) or cancer stem cells (CSCs) in that each tumor formation is governed by a rare subpopulation of cells with self-renewal capacity. CICs have been demonstrated to have capacities of promoting tumor growth, tumor regeneration and metastatic progression. Methods: Cancer cells with Tid1 knockdown or overexpression were generated to evaluate the different properties of stemness and tumorigenicity in vitro and in vivo. Further, the differential gene expression profile of EMT signature and stemness of cancer cell with Tid1 upregulation or downregulation were examined. Results: We observed that Tid1 knockdown enhanced expression of EMT-activator, Snail, and stemness marker Grp78, Nanog, and also exhibited higher ability of migration, invasion, anchorage-independent and sphere formation. In addition, Tid1 overexpression cells decreased cell mobility, sphere formation ability and displayed loss of EMT-activator and stemness marker. Conclusion: Overall, we proved that expression of Tid1 is negatively correlated with EMT and cancer initiation properties in HNSCC. Tid1 potentially could be a key regulator involved in differentiation of cancer cell. Further, therapeutic strategies on target Tid1 mediated malignant properties will be an alternative treatment on for HNSCC. 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 3992. doi:1538-7445.AM2012-3992

Abstract 3385A: Synergistic effect of purified components from Antrodia Cinnamomea Mycelia on targeting oral cancer stem cell

Background: Cancer stem cells (CSCs), also termed as Cancer initiating cells (CICs), represent a rare subpopulation of cells, which are responsible for tumor growth. CICs such as oral cancer -cancer initiating cells (OC-CICs), are often resistant to either radio- or chemotherapy. Therefore, development of drug candidates that specifically target OC-CICs will be effective for future oral cancer therapy. Methods: In this study, we used the in vitro cell-based ALDH activity assay system, which has been demonstrated as a CICs marker, to screen for the active purified components from Antrodia Cinnamomea Mycelia extract (ACME), which can target OC-CICs. In combination with the active components from ACME that targeting OC-CICs, we evaluated the synergistic effect of above active components from ACMS on target oral cancer stem cells. Results: We first found ACME purified compounds (CH-190-WS-A and CH-190-WS-D) can significantly down regulate the ALDH activity of oral cancer cells or OC-CICs, respectively. Treatment of CH-190-WS-A and CH-190-WS-D significantly induced cell death and enhanced the differentiation capability of OC-CICs. Moreover, we found that treatment of CH-190-WS-A and CH-190-WS-D reduced the self-renewal ability, tumorigenic properties and stemness properties of OC-CICs in vitro. Additionally, treatment with CH-190-WS-A or CH-190-WS-D suppressed tumor growth of nude mice bearing tumor xenografts, respectively. Furthermore, treatment of CH-190-WS-D results in down-regulation of mTOR/PI3K/pAKT molecular mechanisms which play an important role in CICs. Whereas treatment of CH-190-WS-A inactivated Notch pathway, which can restore AKT activity then overcome cell death. Finally, combined treatment of CH-190-WS-D and CH-190-WS-A also dramatically caused cell death of OC-CICs through dual-pathway blockage. Conclusion: Our studies reveal combination of ACME purified components can target OC-CICs. Eventually, we can develop therapeutic protocol for alternative oral cancer treatment. 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 3385A. doi:1538-7445.AM2012-3385A

Abstract 2699: Identification and characterization of active components of antrodia cinnamomea mycelia on targeting cancer initiating cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBackground: Cancer initiating cells (CICs) are a subtype of cancer cells, which have the enhanced stemness properties and tumorigenic abilities. CICs are also considered to render resistance of chemotherapy, then, result in recurrence and metastasis. Recent researches and our discovery show that elevated activity of aldehyde dehydrogenase (ALDH) is closely related to CICs. Development of drugs that can specific target cancer initiating cells as a therapeutic method to against the disease would benefit for treatment of cancer patients.nnMethods: We first used the ALDH assay to screen for purified compounds of the myceluim from Antrodia Cinnamomea with inhibitory activity in cancer cells. Then, we find out 4-acetylantroquinonol B (4-AAQN B) significantly down regulated the ALDH activity after its administration. Further, the anticancer activity of 4-AAQN B by targeting CICs will be identified both in vitro and in vivo.nnResults: Our results showed that ALDH activity and stemness marker CD44, uPAR, CCN1 significantly decreased after 4-AAQN B administration. We also observed the decreased cell viability in cancer cells. On note, 4-AQQN B reduced ROS high cells, which are considered as activated CICs.nnConclusion: The anticancer activity of has been reported before. However, we are the first group to confirm that 4-AAQN B can selectively target CICs. The abovementioned findings provide an alternative therapeutics for future cancer treatment in combination with conventional chemotherapy.nnCitation Format: Meng-Chia Wu, Jeng-Fan Lo. Identification and characterization of active components of antrodia cinnamomea mycelia on targeting cancer initiating cells. [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 2699. doi:10.1158/1538-7445.AM2014-2699

Abstract 2099: Tid1 regulates galectin-7 mediated tumorigenesis of oral squamous cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnBackground: We previously identified Tid1, a DnaJ cochaperon protein, functions as a tumour suppressor in oral squamous cell carcinoma (OSCC) tumourigenesis (J Pathol 2009;219:347-55). To further understand the molecular mechanisms mediated by Tid1 on oral tumorigenesis, we applied affinity chromatography and systemic proteomics analysis to identify galectin-7, which specifically interacted with Tid1. Others have shown that Galectin-7 plays a number of important roles in tumor development. However, the role of Galectin-7 in OSCC tumourigenesis remains unclear. Herein, we elucidated the mechanism mediated by Tid1 on regulating galectin-7 mediated cellular signaling and tumorigenesis in OSCC.nnMethods: First, through HA-tag affinity chromatography and mass spectrometry we identified substrate proteins that specifically interact with Tid1. Secondly, co-immunoprecipitation analysis and confocal microscope was performed to confirm the protein-protein interaction between galectin-7 and Tid1. In vitro tumorigenic properties of OSCC overexpressing galectin-7 were determined. Subsequently, the tumorigenic properties of OSCC after co-overexpression of galectin-7 and Tid1 were investigated. Finally, immunoprecipitation analysis was performed to recognize the Tid1 induced deregulation of galectin-7 by enhancing ubiquitination.nnResults: We found out that Tid1 interacted with galectin-7. Furthermore, overexpression galectin-7 expression resulted in up-regulation migration, invasion and soft agar colony formation abilities in OSCC. Importantly, co-overexpression of Tid1 decreased the galectin-7 expression and impaired galectin-7-mediated migration, invasion and soft agar colony formation ability of OSCC. Finally, we found galectin-7 ubiquitination as revealed by antiubiquitin immunoblot analysis in immunoprecipitates.nnConclusion: Our studies first pointed out that galectin-7 played pivotal roles in OSCC tumorigenesis. Notably, we identified galectin-7 that specifically interacted with Tid1. We hypothesized that Tid1/ Hsp70 may regulate galectin-7 stability by ubiquitinylation. The molecular mechanism by which Tid1 negatively modulates the galectin-7 warrants further investigation.nnCitation 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 2099. doi:10.1158/1538-7445.AM2011-2099

Abstract 4382: Matricellular protein Cyr61/CCN1 regulates the stemness of oral cancer stem-like cells

Background: Cysteine-rich 61 (Cyr61 [CCN1]), a matricellular and plasma membrane protein that has disparate function dependent on cell type-specific manner, is closely related to proliferation, invasion, angiogenesis and epithelial-mesenchymal transition (EMT). Besides, EMT promotes stemness properties in normal and neoplastic cells. Therefore, we want to examine whether CCN1 plays a role in our previous enriched oral cancer stem-like cells (OC-SLCs). Methods: Initially, the expression of CCN1 in OC-SLCs translationally was evaluated. Further, the cell surface CCN1 positive cells from oral cancer cells or OC-SLCs were sorted for further study. We will compare the difference of stemness properties and tumorigenicity between CCN1 positive and negative cells. Further, downregulation of CCN1 in oral cancer cells was achieved to evaluate the function of CCN1 on oral cancer tumorigenesis in vitro and in vivo. The molecular mechanisms mediated by CCN1 on regulating the oral cancer tumorigenicity will also be elucidated Results: We found that CCN1 protein was upregulated in OC-SLCs. Comparing to CCN1 negative cells, CCN1 positive cells exhibited higher sphere formation ability and highly expressed stem cell markers, Nanog and Oct-4. Flow cytometry analyses showed that cytoplasmic membrane CCN1 positive OC-SLCs also co-expression with cancer stem-like cell makers, ABCG2, Grp78 and CD133. In addition, CCN1 knockdown cells decreased sphere formation ability and displayed morphological alternation with loss of EMT. Analysis of CCN1 knockdown cells found that EMT related markers were significantly changed. Overall, we demonstrated that CCN1 is involved in maintenance of stemness and acquisition of epithelial-mesenchymal transition in OC-SLCs. Conclusion: Our data suggest that Cyr61 could be a potential oral cancer stem cell marker, and also the key molecule to regulate the stemness of oral cancer stem-like cells. 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 4382. doi:10.1158/1538-7445.AM2011-4382