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

Abstract

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.

ISG15 Is a Critical Microenvironmental Factor for Pancreatic Cancer Stem Cells

“…2A). Of the 19 ISGs detected, we focused on ISG15 as a gene of potential interest as it encodes for a protein that can function intracellularly to modify cytoplasmic and nuclear proteins, it can also be secreted from activated cells as free ISG15, and ISG15 has been shown to play a putative role in other solid tumors such as bladder, oral, prostate, and breast cancers (19)(20)(21)(22)(23)(24). We confirmed the microarray results by RTqPCR and Western blot analysis.…”

“…This apparent overexpression was not all that surprising as Kras-transformed tumors have been shown to overexpress IFNb (38), a strong inducer of ISG15, and our IFNb ELISA and VSV anti-viral assays both confirmed that PDAC cells produce soluble and biologically active IFNb. Interestingly, a growing body of evidence over the past few years has shown a link between ISG15 and tumorigenesis for several solid tumors (19)(20)(21)(22)(23)(24). The general conclusion from these studies is that ISG15 is overexpressed in many tumor cell lines and ISGylation is important for malignant transformation; however, the mechanism(s) by which ISG15 exerts its protumor effects and whether ISG15 functions the same in all solid cancer entities is still unknown.…”

“…For example, while some reports suggest that like ubiquitylation, ISGylation may function in protein turnover (18), it may also play a previously unrecognized role in protein stability (17). The latter has been explored in systems of bladder, oral, prostate, and breast cancers, where high levels of ISG15 and its conjugates have been detected, suggesting a link between ISG15 and tumorigenesis (19)(20)(21)(22)(23)(24). For example, Kiessling and colleagues have shown that in prostate cancer, overexpression of UbE1L increased androgen receptor levels in an ISG15-dependent manner, implying that ISGylation promotes androgen receptor overexpression in cancer cells.…”

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation

“…In vitro cell line-based studies have revealed contrary roles for protein ISGylation in tumorigenesis (27)(28)(29). However, these conclusions depend largely on cell-autonomous events, such as cell proliferation, cell death, and cell evasion.…”

“…Protein ISGylation is strongly enhanced upon pathogen infection and cellular stress related o IFN induction (25). Several in vitro studies using cell lines and human cancer samples have begun to reveal anti-and protumorigenic roles of ISGylation (27)(28)(29). However, in vivo studies of protein ISGylation in tumorigenesis are relatively rare and have become a pressing need for understanding the role of ISG15 in malignant progression.…”