Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C
Alexia T. Kedves, Scott Gleim, Xiaoyou Liang, Dennis M. Bonal, Frederic Sigoillot, Fred Harbinski, Sneha Sanghavi, Christina Benander, Elizabeth George, Prafulla C. Gokhale, Quang-De Nguyen, Paul T. Kirschmeier, Robert J. Distel, Jeremy Jenkins, Michael S. Goldberg, William C. Forrester
Alexia T. Kedves, Scott Gleim, Xiaoyou Liang, Dennis M. Bonal, Frederic Sigoillot, Fred Harbinski, Sneha Sanghavi, Christina Benander, Elizabeth George, Prafulla C. Gokhale, Quang-De Nguyen, Paul T. Kirschmeier, Robert J. Distel, Jeremy Jenkins, Michael S. Goldberg, William C. Forrester
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Research Article Genetics Oncology

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C

Abstract

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

Authors

Alexia T. Kedves, Scott Gleim, Xiaoyou Liang, Dennis M. Bonal, Frederic Sigoillot, Fred Harbinski, Sneha Sanghavi, Christina Benander, Elizabeth George, Prafulla C. Gokhale, Quang-De Nguyen, Paul T. Kirschmeier, Robert J. Distel, Jeremy Jenkins, Michael S. Goldberg, William C. Forrester

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Figure 7

Knockdown of UBC in UBBLO cells causes a ubiquitin depletion catastrophe.

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Knockdown of UBC in UBBLO cells causes a ubiquitin depletion catastrophe...
(A) Viability effects at 72 hours (left) in UBBWT (A2058, black) or UBBLO (HMCB, red) melanoma cells transduced with a doxycycline-inducible (Dox-inducible) shUBC hairpin and exposed to a dose titration of Dox. Studies were performed twice and analysis is replicates with SEM. Knockdown of UBC mRNA is shown at right (48 hours after Dox) and was performed twice with technical replicates and SEM. (B) HMCB (UBBLO) cells with shUBC or nontargeting hairpin control shNT were treated with 100 ng/ml Dox, and protein lysates were prepared at the time of Dox addition and at 12, 24, 36, and 48 hours thereafter. Antibodies used for Western blotting were against ubiquitin, γ-H2AX, c-myc, TP53, total H2AX, and β-actin. Molecular weight markers (kDa) are shown on the left. (C) Dox (100 ng/ml) was added to cells and lysates prepared at indicated times. Cycloheximide (CHX) was added (10 and 50 μg/ml) 6 hours prior to harvesting. Analysis of protein lysates following Dox time course was performed twice each with samples prepared from independent experiments.