Changes in Tumor Growth and Metastatic Capacities of J82 Human Bladder Cancer Cells Suppressed by Down-regulation of Calreticulin Expression Speaker: Yi-Chien Lu Institute of Zoology, National Taiwan University

Contents  Introduction & Rationale  Results  Conclusion

 One of the main leading causes of cancer related death. (15 th in Taiwan ; 8 th in USA) Bladder Cancer Wockhardt Hospital Blog  Transitional cell carcinoma (TCC), a malignancy which grows from epithelium.

 30% of patients present with a muscle-invasive metastasis and 85% of these patients will die from the disease within 2 yr. Bladder Cancer and Metastasis Nature Reviews Cancer 3, (January 2003) 1. In situ cancer 4. Invasion of the circulation system: survival, transport 2. Invasion of the tumor border 3. Lymphatic spread 6. Solitary dormant cells occult micrometastasis 7. Progressive colonization angiogenesis 5. Arrest extravasion Invasion Migration Adhesion Proliferation

Kageyama S. er al. International Journal of Urology (2009) Biomarker for bladder urothelial carcinoma - Urinary Calreticulin. Bladder cancer Non-bladder cancer Positive correlation with the histological grade and pathologic T stage.

Biochem. J. (2009) 417, 651–666  46 kDa  Ca 2+ homeostasis  chaperone in the ER  adhesion, migration, and gene expression. What is Calreticulin (CRT)?

Calreticulin in different cancers  In some cancers, tumor tissues express higher levels of CRT compared with normal tissues. Hepatoma ( Ding SJ et al. 2004) Prostate cancer (Alaiya A et al., 2000) Colon cancer (Alfonso P e al., 2005) Vaginal cancer (Hellman K et al., 2004)  Overexpression of CRT increases cell proliferation, migration, and upregulates VEGF and PlGF in gastric cancer. CN Chen et al. (2009) Calreticulin in different cancers

Rationale To investigate the roles of CRT in bladder cancer progression.  CRT might play a crucial role in cancer progression.  CRT was increased in urinary cancer tissue and urinary CRT was proposed as a biomarker for bladder cancer.

Bladder cancer cell Hypothesis CRT Proliferation, Migration, & Adhesion Tumor growth & Metastasis

Results

CRT Actin mRNA Protein expression Real-time PCR (A) and Western Blot (B) show that mRNA and protein expression in calreticulin stable cell line.

J82 (bladder cancer cell) Select stable cell lines Cell cycle analysis Chemotasis assay Adhesion assay Cell behaviors Animal model in vivo in vitro

A. Knockdown of calreticulin suppresses bladder cancer cell proliferation.

Down-regulation of CRT inhibited bladder cancer proliferation through affecting cell cycle transition rather than apoptosis.

Cell adhesion and migration ability were regulated by CRT expression levels. Adhesion Migration

J82 (bladder cancer cell) Select stable cell lines Cell behaviors Animal model in vivo in vitro Cell cycle analysis Chemotasis assay Adhesion assay Subcutaneous injection model Tail vein injection for metastasis

Knockdown of CRT diminished tumor formation in nude mice. PCR3.1 CRT-RNAi

Knockdown of CRT inhibited metastasis of bladder cancer cells.

Tail vein injection

Liver metastases were only observed in control group.

Lung metastases were observed in both group.

Knockdown of CRT inhibited metastasis of bladder cancer cells.

Bladder cancer cell Summary CRT Migration & Adhesion Cancer Metastasis Proliferation Tumor Growth

Acknowledgment Institute of Zoology, NTU Bojeng Wang (Institute of Zoology) Lab504 Advisers: Hsinyu Lee (Institute of Zoology & Life Science, NTU) Cheng-Chi Chang (Graduate Institute of Oral Biology, NTU) * Grant supported by 98R0318 from National Taiwan University. National Taiwan University Hospital Chiung-Nien Chen Wen-Ming Hsu Szu-Ta Chen

Thank you for attention !!