Studies of the Proteasome Inhibitor Sensitivity Modulator Tight Junction Protein 1 Highlight a Role for Signaling through the Epidermal Growth Factor Receptor in Determining Proteasome Capacity
Authors: Zhang Xing-Ding , Baladandayuthapani Veerabhadran , Lin Heather Yan , Mulligan George , Li Bin , Esseltine Dixie-Lee , Qi Lin , Xu Jian-Liang , Hunziker Walter , Barlogie Bart , Usmani Saad Z. , Zhang Qing , Crowley John , Hoering Antje , Li Bing-Zong , Wang Huihan , Zhang Jiexin , Kuiatse Isere , Tang Jin-Le , He Jin , Yang Jing , Davis Richard Eric , Ma Wencai , Wang Zhiqiang , Yang Lin , Orlowski Robert Z. 
Published: November 2019

Abstract
Background:
Proteasome inhibitors (PIs) are important agents against myeloma, but innate and acquired resistance limit their effectiveness. We previously found Tight junction protein (TJP)-1 to be a modulator and potential biomarker of PI sensitivity, via studies of bortezomib resistant models and clinically annotated gene expression profiling (GEP) databases of patient samples. Also, we showed that its expression reduced EGFR/JAK1/STAT3 pathway activation and proteasome capacity. However, the molecular mechanisms by which TJP1 expression suppressed EGFR signaling and enhanced PI sensitivity were not understood.
Methods:
We performed co-immunoprecipitation (co-IP) studies to understand interactions of TJP1 with EGFR, and evaluated the impact of various cytokines on proteasome capacity. Also, genetic and pharmacologic approaches were used to modulate EGFR/JAK/STAT activity to determine the effects on PI sensitivity. Finally, gene set enrichment analyses (GSEA) were performed to determine if EGFR activation signatures could be identified in primary samples, and if they correlated with outcomes.
Results:
Previous studies of TJP1 focused on myeloma models, but since bortezomib is also approved for mantle cell lymphoma, we sought to determine if TJP1 influenced sensitivity here as well. Suppression of TJP1 using shRNAs reduced the sensitivity of JeKo-1 and MINO cells to bortezomib. In addition, murine embryonic stem cells (mESCs) with TJP1 knockout were more resistant to PIs than was the case for wild-type mESCs. Moreover, the TJP1 knockout mESCs expressed higher levels of both activated EGFR and immunoproteasome subunits PSMB8 and 9, and had greater levels of proteasome chymotrypsin-like (ChT-L) activity. Also, re-introduction of TJP1 into the knockout mESCs ehanced their PI sensitivity, and reduced the ChT-L activity. Co-IP studies showed that IP of TJP1 also led to detection of EGFR, and vice versa. Phospho-specific antibodies showed that TJP1 precipitated predominantly the non-phosphorylated, inactive EGFR. Indeed, when cells were treated with EGF and extracts were subjected to IP for TJP1, reduced levels of EGFR were precipitated. Evaluating other receptor tyrosine kinases, we found that TJP1 precipitated IGF-1R but not IL-6R. Therefore, we treated myeloma cells with EGF, IL-6, or IGF-1; all reduced PI sensitivity, but only EGF enhanced proteasome ChT-L activity and capacity. Moreover, shRNA-mediated EGFR knockdown, or pharmacologic inhibition with erlotininb in myeloma cells reduced JAK1/STAT3 activity, levels of PSMB8 and 9, and ChT-L activity, and enhanced the activity of bortezomib. Since the PSMB8 and PSMB9 promoters have consensus STAT3 binding sites, we knocked down STAT3 and found that this also reduced PSMB8 and 9 and ChT-L activity, and enhanced the efficacy of bortezomib. In a mouse model of lytic bone destruction by xenografted RPMI 8226 cells, the ability of bortezomib to increase bone trabecular volume was blunted by TJP1 suppression, consistent with a lower anti-myeloma effect. Lastly, we used publically available EGF-stimulated and EGFR inhibitor gene signatures to examine the clinically annotated GEP data from myeloma patients. This showed that: 1) TJP1 expression in myeloma samples from patients on the University of Arkansas Total Therapy studies was positively correlated with known signatures of inhibitors of EGFR signaling; 2) in GSEA, signatures of EGF and EGFR signaling were significantly positively enriched when genes were ranked by correlation with TJP1; and 3) an EGFR-related gene set had significant negative correlation with survival duration in the Total Therapy 3 dataset.
Conclusions:
Taken together, our data support the role of TJP1 as a negative regulator of EGFR signaling through its ability to specifically bind, and possibly stabilize the non-phosphorylated form of EGFR. Furthermore, they demonstrate that TJP1 influences proteasome capacity through EGFR/JAK/STAT signaling, and that approaches to suppress this pathway hold promise to overcome PI resistance and achieve PI sensitization. Finally, they validate a heretofore underappreciated role for EGF/EGFR signaling in myeloma pathobiology and patient outcomes.

Disclosures
Mulligan: Millennium Pharmaceuticals: Employment. Usmani:Celgene Corporation: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy; Array BioPharma: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding. Orlowski:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Research Funding; JW Pharmaceutical: Research Funding; Array BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.