American Association for Cancer Research
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Supplementary Tables 1 and 2 and Supplementary Figures 1 through 6 from Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation

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posted on 2023-03-31, 00:25 authored by Wayne O. Miles, Antonio Lembo, Angela Volorio, Elena Brachtel, Bin Tian, Dennis Sgroi, Paolo Provero, Nicholas Dyson

Supp Table 1: APA effected PUM target in breast tumors Supp Table 2: primers and siRNAs used Supp Fig 1: TNBC tumor 3'UTR RT-PCR, FOXO1, E2F4 and PTEN Supp Fig 2: TNBC tumors 3'UTR RT-PCR NRAS Supp Fig 3: TNBC tumors 3'UTR RT-PCR c-JUN Supp Fig 4: NRAS, PTEN and FOXO1 regulation in multiple cell lines Supp Fig 5: NRAS and c-JUN stability assays Supp Fig 6: Regulation of 3'UTR length

Funding

NIH

Avon Foundation

DOD

Susan G. Komen for the Cure

MGH

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ARTICLE ABSTRACT

Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3′ untranslated region (3′ UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3′ UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype–specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. Cancer Res; 76(24); 7231–41. ©2016 AACR.