Dr. Eri Srivatsan has focused his research on cervical cancer, which is the second most common cancer responsible for cancer-related death in women around the world. The incidence is increasing, with 450,000 new cases diagnosed annually worldwide. The disease is frequently found in women having multiple sex partners, smoking habits and immune system dysfunctions. Although HPVs (Human Papilloma Viruses) play an important role in tumor development, detailed studies have shown that viral infection is not sufficient and additional risk factors, including genomic alterations, are involved. Researchers have now identified a tumor suppressor gene, cystatin E/M, which may be involved in the control of cervical cancer development.
The researchers have previously used microsatellite analysis of cervical cancer cell lines and primary tumors to localize a tumor suppressor gene to a 300 kb interval of chromosome 11q13. Of the 11 genes mapped to this 300 kb interval, cystatin E/M, a cysteine protease inhibitor, seemed to be a potential candidate due to reports of its involvement in breast cancer. Researchers have now obtained evidence confirming cystatin E/M as the cervical cancer suppressor gene.
Both the RT-PCR (reverse transcriptase PCR) and western blotting studies revealed absence of gene expression in six different cervical cancer cell lines and in nine of 11 primary tumors. Examination of the three exons in 19 primary tumors has revealed exonic mutation in six tumors including homozygous deletion (complete loss of sequences from both the alleles) of exon 1 in a tumor. In four of the cell lines and a primary tumor, loss of expression also correlated to promoter hypermethylation. Re-expression of the gene in two different cell lines after treatment with the demethylating agent 5-aza deoxycytidine confirmed the presence of promoter hypermethylation. Ectopic expression of the gene in the cell lines resulted in cell growth inhibition and apoptosis in vitro and inhibition of tumor growth in vivo in nude mice. Srivatsan’s studies further revealed that cathepsin L, a protein overexpressed in a number of tumors and linked to invasion and metastasis as the physiological target of cystatin E/M. Finally, silencing of cathepsin L led to decreased cell growth and over expression promoted cell growth. Thus, his results clearly demonstrated that cystain E/M is a cervical cancer suppressor gene and controls cell growth through the inhibition of cathepsin L. Expression analysis of normal skin, cervical and endocervical tissues, 30 different primary tumors and 30 different CINs (cervical intraneoplasias) by immunohistochemistry have shown expression of cystatin E/M in the normal tissues and CINs. Absence of gene expression in primary tumors clearly indicated a role for cystatin E/M in tumor development. Expression of cathepsin L, on the other hand, was only observed in the tumors.
Cystatin E/M protein is a secreted protein and therefore Srivatsan analyzed cervical cancer cell line (HeLa cell) growth with the addition of cell free supernatant from cystatin E/M expressing cells. Srivatsan could observe growth suppression indicating non cell autonomous growth inhibition. He has also performed Boyd chamber studies where the growth factors secreted by the cells in the inset well could influence the growth of cells outside the Boyd chamber. Again, he could observe cell growth suppression confirming non cell autonomous growth inhibition by the suppressor gene. Finally, he has evidence to suggest inactivation of cystatin E/M by the E7 protein of HPV 16 indicating a HPV mediated inactivation of the suppressor gene, similar to the inactivation of p53 and Rb proteins by the E6 and E7 proteins of HPV.
He has created a lentiviral tetracycline inducible system where he was able to induce the expression of cystatin E/M gene with the addition of doxycyclin (analog of the antibiotic tetracycline) into the culture medium or in the drinking water in nude mouse studies. The in vitro studies have shown growth suppression of the HeLa cells with the induction of the cystatin E/M gene. He has also observed non-cell autonomous growth inhibition of the HeLa cells using supernatant and boyden chamber studies. The expressed cystatin E/M reduces the expression of the substrate cathepsin L both in the cells and in the secreted media supernatant indicating the mechanism of cell growth inhibition. He has further observed non-cell autonomous growth inhibition of multiple myeloma cells by the cystatin E/M gene induced in the HeLa cells in co-culture studies.
Expression analysis of normal skin, cervical and endocervical tissues, 35 different primary tumors and 15 different CINs (cervical intraneoplasias) by immunohistochemistry have shown expression of cystatin E/M in the normal tissues and CINs. Absence of gene expression in primary tumors clearly indicated a role for cystatin E/M in tumor development. Expression of cathepsin L on the other hand was observed only in the tumors. He now plans to determine the molecular mechanism of cystatin E/M mediated inhibition of cathepsin L and its role in tumor suppression using the inducible vector system.
Srivatsan also has an active research program on the molecular mechanism of cisplatin resistance in head and neck cancer. Here, he is trying to determine the role of curcumin, the active ingredient of the Indian spice turmeric, as an adjuvant therapy for head and neck cancer.
Selected Cancer-Related Publications:
Rayess H, Wang MB, Srivatsan ES. Cellular senescence and tumor suppressor gene p16. Int J Cancer. 2012 Apr 15;130(8):1715-25. doi: 10.1002/ijc.27316. Epub 2011 Dec 5.
Kim SG, Veena MS, Basak SK, Han E, Tajima T, Gjertson DW, Starr J, Eidelman O, Pollard HB, Srivastava M, Srivatsan ES, Wang MB. Curcumin treatment suppresses IKKBeta kinase activity of salivary cells of patients with head and neck cancer: a pilot study. Clin Cancer Res. 2011 Sep 15;17(18):5953-61. Epub 2011 Aug 5.
Wilken R, Veena MS, Wang MB, Srivatsan ES. Curcumin: A review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma. Mol Cancer. 2011 Feb 7;10:12.
Duarte VM, Han E, Veena MS, Salvado A, Suh JD, Liang LJ, Faull KF, Srivatsan ES, Wang MB. Curcumin enhances the effect of cisplatin in suppression of head and neck squamous cell carcinoma via inhibition of IKKBeta protein of the NFKappaB pathway. Mol Cancer Ther. 2010 Oct;9(10):2665-75.
Basak SK, Veena MS, Oh S, Huang G, Srivatsan E, Huang M, Sharma S, Batra RK. The malignant pleural effusion as a model to investigate intratumoral heterogeneity in lung cancer. PLoS One. 2009 Jun 12;4(6):e5884.