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Identification and Characterization of Cancer Stem Cells in Hepatocellular Carcinoma

Identification and Characterization of Cancer Stem Cells in Hepatocellular Carcinoma Xin-yuan Guan Department of Clinical Oncology The University of Hong Kong. Hepatocellular Carcinoma (HCC). HCC 5 th most common cancer 3 rd leading cause of cancer death

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Identification and Characterization of Cancer Stem Cells in Hepatocellular Carcinoma

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  1. Identification and Characterization of Cancer Stem Cells in Hepatocellular Carcinoma Xin-yuan Guan Department of Clinical Oncology The University of Hong Kong

  2. Hepatocellular Carcinoma (HCC) HCC • 5th most common cancer • 3rd leading cause of cancer death • High incidence geographic regions: Southeast Asia, China, sub-Saharan Africa Etiology of HCC • HBV or HCV infection • Aflatoxin B1 intake • Alcohol abuse • Obesity, diabetes and nonalcoholic fatty liver disease

  3. Tumor-initiating cells (TICs) / Cancer stem cells (CSCs) • CSC has been defined as a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells. • Properties of CSCs – persistent self-renewal, sustained proliferation, tumor initiation. • Other common (but not defining) characteristics of CSCs – rarity within a tumor, stem cell marker expression, ability to differentiate along multiple lineages, chemoresistance, metastasis, etc. • Origin of CSCs – largely unknown. • e.g. for leukemia and colon cancer – normal stem cells • e.g. for liver cancer – ?

  4. Identificating CD133as a specific CSC surface marker in HCC CD133 / Prominin-1 Partial hepatectomy mouse model cDNA microarray before vs after PH Mouse – Severe Partial Hepatectomy ‘Inactive’ Active Regeneration Restored CD133 - ++ + day 0 day 3 day 7 Time following PH Ma S, et al. Gastroenterology 2007

  5. CD133+ cells in some of cultured human liver cell lines Cell line Characteristics CD133+(%) Tumorigenicity MIHA immortalized, normal 0 N H2-P primary HCC 4 N H2-M metastatic HCC 6 N H4M metastatic HCC 0 N HepG2 hepatoblastoma 8 N QGY7701 HCC 0 N BEL7402 HCC 1 N Huh7 HCC 45-65 Y PLC8024 HCC 50-60 Y Ma S, et al. Gastroenterology 2007

  6. CD133 expression in human HCC clinical specimens CD133+ cell could be detected in almost all primary HCCs ranged from 1% to 13%. Ma S, et al. Cell Stem Cell 2010

  7. Characteristics of CD133+ subpopulation in HCC • CD133+ cells exhibit increased tumorigenic potential • CD133+ cells exhibit an increased potential for self-renewal • CD133+ cells exhibit a marked ability to differentiate • CD133+ cells preferentially express “stemness” genes • CD133+ cells are more resistant to anticancer agents, doxorubicin and 5-fluorouracil

  8. in vivo animal models CD133- CD133+ * * CD133- CD133+ CD133+ 400 CD133- p = 0.003 p < 0.001 8 CD133- 300 Clonogenicity (%) Number of colonies 6 200 100 4 CD133+ 0 2 CD133- CD133+ 1 2 subcutaneous (nude) intra-hepatic CD133- CD133+ 0 1 2 CD133+ HCC cells exhibit increased tumorigenic potential CD133- CD133+ Soft Agar Colony Formation Assay 5 x 104 cells Proliferation Assay subcutaneous (SCID) 1 x 103 cells Ma S, et al. Gastroenterology 2007

  9. CD133+ cells exhibit an increased potential forself-renewal Serial transplant of cells re-sorted from xenograft tumors induced by CD133+ cells into secondary mice recipients 1. Tumor incidence and latency in secondary recipients were similar to those of primary xenograft tumors. 2. Transplanted CD133+ cells can give rise to a heterogeneous tumor.

  10. CD133+ HCC cells exhibit increased potential for differentiation CD133+ and CD133- cells were cultured in M199 medium with VEGF, BDNF and bFGF. CD133- after differentiation CD133+ after differentiation CD133+ cells differentiate to produce non-hepatocyte-like, angiomyogenic-like cells in vitro Ma S, et al. Gastroenterology 2007

  11. CD133+ HCC cells preferentially express “stemness” genes CD133+ (Huh7) xenograft tumor PLC8024 Huh7 CD133 + - C + - C + - C CD133 β-catenin Oct-3/4 Bmi SMO Notch-1 18S RNA Pathways Implicated in the Self-renewal and Differentiation of Stem Cells: Wnt/β-catenin, Notch, Hedgehog/SMO, Bmi and Oct3/4 Ma S, et al. Gastroenterology 2007

  12. CD133+ HCC cells confer chemoresistance via the preferential activation of the Akt/PKB and Bcl-2 survival pathway Cell Proliferation Assay CD133+ cells preferentially expressed survival proteins P-Akt, P-Bad, Bcl-2 Ma S, et al. Oncogene 2008

  13. Inactivation of Akt/PKB pathway by an inhibitor to the pathway abolishes the survival of CD133+ cells Western Blot Cell Proliferation Assay Ma S, et al. Oncogene 2008

  14. CD133 as a prognostic marker for HCC patients Clinicopathological correlation of CD133 with HCC Increased CD133 expression in HCC is correlated with advanced disease stage and worse overall survival. Ma S, et al. Cell Stem Cell 2010

  15. up-regulated miRNAs miR-130b promotes CD133+ liver CSC growth and self-renewal via TP53INP1 HCC patient clinical specimens dataset (5 patient sets) miR-130b is preferentially expressed in CD133+ CSCs HCC cell lines dataset (PLC8024 and Huh7) miRNA profiling of CD133+ CSCs and CD133- cell counterparts miR-130b miR-369-5p miR-19a miR-369-3p miR-138 miR-302a miR-200b miR-146a miR-181a miR-181b miR-181d miR-197 miR-223 miR-125b miR-135a miR-218 miR-219 miR-24 miR-150 miR-199a let-7b miR-26b miR-93 miR-126 miR-142-3p let-7a miR-30c miR-33 miR-153 miR-1 miR-10b miR-130b exhibits an increased ability to self-renew, initiate tumors and resist chemotherapy miR-368 miR-372 miR-373 down-regulated miRNAs Ma S, et al. Cell Stem Cell 2010 Ma S and Guan XY. Cell Cycle 2011

  16. TP53INP1 is a direct functional target of miR-130b Expression of TP53INP1 is negatively correlated with CD133 and miR-130b Ma S, et al. Cell Stem Cell 2010 Ma S and Guan XY. Cell Cycle 2011

  17. TP53INP1 regulates tumor formation and self-renewal in CD133+ liver CSCs TP53INP1 was silenced in CD133- PLC8024 cells Ma S, et al. Cell Stem Cell 2010 Ma S and Guan XY. Cell Cycle 2011

  18. Summary Characteristics of CD133+ subpopulations, isolated from HCC clinical specimens and cell lines are consistent with the predicted behavior of cancer stem cells. Ma et al., Gastroenterology 2007 CD133+ CSCs contribute to HCC chemoresistance through activation of the specific Akt/PKB and Bcl-2 survival pathway. Ma et al., Oncogene 2008 Aldehyde dehydrogenase (ALDH) is preferentially expressed in CD133+ cells and can be used to better characterize the CD133+ HCC CSC population. Ma et al., Mole Cancer Res 2008 miR-130b is overexpressed in CD133+ CSCs and regulates tumor growth and self-renewal via tumor protein TP53-INP1. Ma et al., Cell Stem Cell 2010 CD133+ liver CSCs promote tumor angiogenesis through activation of the NTS / IL-8 / CXCL1 signaling cascade via MAPK signaling. Tang KH, et al. Hepatology 2011 Knowckdown of CD133 can inhibit tumorigenicity of HCC cell lines. Tang KH, et al. Hepatology 2011

  19. Acknowledgments Department of Clinical Oncology, HKU Department of Surgery, HKUDr. Stephanie Ma Dr. Kwan Ho Tang Prof. Chung Mau Lo Ms. Yangyang Song Dr. Polly Chen Dr. Kwan MAN Ms. Carol Tong Mr. Pak Shing Kwan Department of Pathology, HKU Dr. Kwok Wah Chan

  20. Hong Kong Thank You Questions or Comments

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