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Disruption of the KEAP1-NRF2 pathway results in the transactivation of NRF2 target genes, consequently inducing cell proliferation and other phenotypic changes in cancer cells. Here, we demonstrated that GULP1 was a KEAP1-binding protein that maintained actin cytoskeleton architecture and helped KEAP1 to sequester NRF2 in the cytoplasm. In urothelial carcinoma of the bladder (UCB), silencing of GULP1 facilitated the nuclear accumulation of NRF2, led to constitutive activation of NRF2 signaling, and conferred resistance to the platinum drug cisplatin. Knockdown of GULP1 in UCB cells promoted tumor cell proliferation in vitro and enhanced tumor growth in vivo. In primary UCB, GULP1 silencing was more prevalent in muscle-invasive UCB compared to nonmuscle-invasive UCB. GULP1 knockdown cells showed resistance to cisplatin treatment. In parallel with decreased GULP1 expression, we observed increased expression of NRF2, HMOX1, and other candidate antioxidant genes in cisplatin-resistant cells. Furthermore, low or no expression of GULP1 was observed in most cisplatin nonresponder cases. Silencing of GULP1 was associated with GULP1 promoter hypermethylation in cell lines and primary tumors, and a high frequency of GULP1 promoter methylation was observed in multiple sets of primary clinical UCB samples. Together, our findings demonstrate that GULP1 is a KEAP1-binding protein that regulates KEAP1-NRF2 signaling in UCB and that promoter hypermethylation of GULP1 is a potential mechanism of GULP1 silencing.

Background: Prospective cohort studies have reported spontaneous regression rates of high-grade cervical intraepithelial Neoplasia (CIN2/3) close to 35%, over a period of 4-6 months. These regressions are presumably immunologically mediated. It is now well recognized that PD-L1 is an immune checkpoint used by neoplastic cells to evade the immune system, particularly T cells that are specific for tumor antigens. PD-L1 and several other immune checkpoints consist of inhibitory pathways that tightly regulate the immune system to control the duration and amplitude of immune responses in peripheral tissues in order to minimize collateral tissue damage. Design: Immunohistochemistry with anti-TBX21 and anti-PD-L1 antibodies was performed in formalin-fixed paraffin-embedded tissue sections from a tissue microarray consisting of invasive cervical carcinomas (ICCs) (n = 22), CIN2/3 (n = 4) and benign cervical tissues (n = 4). Expression of TBX21 and PD-L1 was reviewed and scored by a trained pathologist blinded to the clinical outcome of each case. Statistical correlations between these markers’ score and available demographic and clinicopathologic data from each case were performed, as well as disease-free survival curves. Moreover, PD-L1 staining was performed in three patients with CIN2/3 that received peripheral (IM) therapeutic HPV vaccination. PD-L1 expression differences in pre and post-vaccination tissues were compared. Results: Increased expression of TBX21+ T-cells and PD-L1 was observed from benign to CIN2/3 to ICCs. Association analysis (fisher exact test) showed a significant correlation between increased intraepithelial and stromal infiltration of TBX21+ T-cells (score 2,3) and higher PD-L1 membrane expression (p = 0.04 and p = 0.005, respectively). No significant associations were found between PD-L1 expression and demographic and clinicopathologic information, likely due to small number of samples. In our vaccine cohort, we observed a stronger membrane PD-L1 expression in the epithelium and stroma of the post-vaccination tissue compared to the pre-vaccination biopsy. Conclusion: Increased membrane expression of PD-L1 is tightly correlated with an increased epithelial and stromal infiltration of TBX21+ T cells in CIN2/3 and ICCs. This correlation was also observed in our vaccination cohort when compared to our previous published findings of immune activation after HPV therapeutic vaccination. This association could represent an immune response control mechanism induced presumably by interferons released by TBX21+ T cells. We are currently evaluating the expression of PD-L1 and B7-H4, a recently discovered inhibitory ligand, in a larger cohort of benign, CIN2/3 and ICCs. Finally, we will further explore, quantitate and compare the expression changes of these immune checkpoints in the remaining CIN2/3 cases of our vaccination cohort (n = 12) in pre and post-vaccination tissues. Citation Format: Leonel F. Maldonado Gonzalez, Christopher VandenBussche, Richard Roden, T.C. Wu, Benjamin Tycko, Cornelia L. Trimble. PD-L1: the hand brake of immune responses in cervical intraepithelial neoplasia and cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1320. doi:10.1158/1538-7445.AM2015-1320