ISGylation governs the oncogenic function of Ki-Ras in breast cancer

Aberrant expression of the oncogenic Kirsten-Ras (Ki-Ras) and interferon-stimulated gene 15 (ISG15) pathways is common in breast and other cancers. However, whether these dysregulated pathways cooperate to promote malignancy is not known. This study links Ki-Ras and ISG15 in a previously unidentified regulatory loop that may underlie malignant transformation of mammary cells. We show that oncogenic Ki-Ras regulates the expression of the ISG15 pathway (free ISG15 and ISG15 conjugates), and ISG15, in turn, stabilizes Ki-Ras protein by inhibiting its targeted degradation via lysosomes in breast cancer cells. Disruption of this loop by silencing either Ki-Ras or the ISG15 pathway restored the disrupted cellular architecture, a hallmark feature of most cancer cells. We also demonstrate that ISG15 and UbcH8 (ISG15-specific conjugating enzyme) shRNAs reversed Ki-Ras mutation-associated phenotypes of cancer cells, such as increased cell proliferation, colony formation, anchorage-independent growth in soft agar, cell migration, and epithelial-mesenchymal transition. As UbcH8-silenced breast cancer cells are devoid of ISG15 conjugates but have free ISG15, our results using UbcH8-silenced cells suggest that ISG15 conjugates, and not free ISG15, contributes to oncogenic Ki-Ras transformation. We have thus identified the conjugated form of ISG15 as a critical downstream mediator of oncogenic Ki-Ras, providing a potential mechanistic link between ISG15 and Ki-Ras-mediated breast tumorigenesis. Our findings, which show that inhibition of the ISGylation reverses the malignant phenotypes of breast cancer cells expressing oncogenic Ki-Ras, support the development of ISG15 conjugation inhibitors for treating breast and also other cancers expressing oncogenic Ki-Ras.