Abstract 2257: Divergent IRE1α endonuclease outputs dictate the senescence response of mouse keratinocytes to oncogenic HRAS

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oncogenic RAS plays a pivotal role in neoplastic transformation, however in primary cells it initially triggers a proliferative response followed by irreversible growth arrest and premature senescence, a potent barrier to tumor development. How premature senescence is bypassed to enable full transformation and tumor progression is not clear. Here we show that in primary mouse keratinocytes IRE1α, an endoplasmic reticulum (ER) transmembrane kinase/endoribonuclease (RNase) important in the unfolded protein response, regulates cell fate in response to oncogenic v-Ha-RAS (HRAS) through its two distinct RNase outputs. During the initial proliferation response, oncogenic HRAS activates IRE1α-mediated cleavage of XBP1 mRNA to produce the active transcription factor that is MEK-ERK dependent. Microarray analysis of keratinocytes with IRE1α knockdown using shRNA also showed that HRAS activates the regulated IRE1α-dependent mRNA decay (RIDD) function of IRE1α and this is responsible for downregulation of a subset of mRNAs by HRAS. Interestingly, IRE1α and XBP1 knockdown had distinct effects on premature senescence in HRAS keratinocytes. XBP1 knockdown induced premature senescence that was associated with extensive vacuolization whereas IRE1α knockdown did not despite similar inhibition of proliferation. Pharmacologic inhibition of IRE1α RNase activity inhibited premature senescence in XBP1-depleted HRAS keratinocytes and enhanced proliferation demonstrating a role for RIDD in this response. In addition, the potent cytostatic growth factor TGFβ1 which we previously showed induces rapid senescence in HRAS keratinocytes, inhibited IRE1α-mediated XBP1 splicing and enhanced RIDD possibly through alteration of IRE1α phosphorylation. Similarly, shRNA knockdown or pharmacologic inhibition of IRE1α bypassed TGFβ1-induced senescence, while XBP1 knockdown was susceptible to it. Using the RIDD gene signature obtained from microarray analysis of HRAS keratinocytes, we performed gene set enrichment analysis (GSEA) on previously identified gene expression profiles of benign and malignant mouse epidermal squamous tumors. Our results, validated by qPCR, show that genes in the RIDD signature are downregulated in benign tumors, paralleling the in vitro studies, but are upregulated in malignant tumors. In contrast, XBP1 splicing is similar in both tumor types. These results suggest that RIDD is linked senescence in vitro and benign tumor phenotype in vivo and that suppression of RIDD, but not XBP1 splicing, is a prerequisite for malignant progression. We propose that the balance between XBP1 splicing and RIDD determines whether a premalignant tumor cell undergoes proliferation and tumor progression or senescence and tumor suppression. Selective pharmacological agents that inhibit XBP1 splicing or enhance RIDD could be an important therapeutic modality to the eradication of cancer. Citation Format: Nicholas Blazanin, Christian John, Alayna Craig-Lucas, Adam Glick. Divergent IRE1α endonuclease outputs dictate the senescence response of mouse keratinocytes to oncogenic HRAS. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2014-2257