Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB

Abstract We and others have shown that cystatin E/M gene is inactivated in primary human tumors pointing to its role as a tumor suppressor gene. However, the molecular mechanism of tumor suppression is not yet understood. Using plasmid directed cystatin E/M gene overexpression, lentiviral mediated tetracycline inducible vector system and HPV 16 E6 and E7 gene immortalized normal human epidermal keratinocytes, we demonstrate intracellular and non-cell autonomous apoptotic growth inhibition of tumor cell lines and that growth inhibition is associated with cytoplasmic retention of NF-κB. We further demonstrate decreased phosphorylation of IKKβ and IκBα in the presence of TNFα confirming the role of cystatin E/M in the regulation of NF-κB signaling pathway. Growth suppression of nude mouse xenograft tumors carrying a tetracycline inducible vector system was observed with the addition of doxycycline in drinking water confirming cystatin E/M as a tumor suppressor gene. Finally, immunohistochemical analysis of cervical carcinoma in situ and primary tumors have shown statistically significant inverse relationship between the expression of cystatin E/M and cathepsin L and direct relationship between the loss of cystatin E/M expression and nuclear expression of NF-κB. We therefore propose that cystatin E/M suppressor gene plays an important role in the regulation of NF-κB.