Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation

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.

Figure 1

PRE-containing mRNAs are disproportionately targeted by alternative polyadenylation. A, Percentage of the 921 mRNAs shortened by alternative polyadenylation in breast tumors that lose PRE (PUM) or miRNA seed sequences (**, P < 0.01). B, Percentage of the 189 mRNAs lengthened by alternative polyadenylation in breast tumors that gain PRE (PUM) or miRNA seed sequences. C, Breast tumor subtype shared and unique APA events. Table, Gene ontology of PRE-containing mRNAs shortened in triple-negative breast tumors.

Figure 2

Candidate gene alternative polyadenylation status in an independent cohort of triple-negative breast tumors. A, RT-PCR of FOXO1 from tumor and control samples. Tumors were subdivided on the basis of FOXO1 length at UTR (>50% or <50%), full length and 3'UTR extended. Black bracket indicates lengthened 3′ UTRs in tumors and the number represents the number of tumors displaying this APA event. B, RT-PCR of PTEN from tumor and control samples. Black bracket, number of shortened 3′ UTRs. C, RT-PCR of NRAS from tumor and control samples. Black bracket, number of shortened 3′ UTRs. Tumors were subdivided on the basis of 3' UTR length (>50% or <50%), short and long. D, RT-PCR of the c-JUN from tumors and control samples. Black bracket, number of shortened 3′ UTRs. E, Percentage of tumors displaying APA of each transcript. E2F4 was used as a negative control. F, NRAS target gene expression (FOXA1 and SMG1) in tumors expressing either the long or short 3′ UTR isoform of NRAS (*, P < 0.05; **, P < 0.01). G, c-JUN target gene expression (KRT7 and TIMP1, activated; KRT19, repressed) in tumors expressing either the long or short 3′ UTR isoform of c-JUN (*, P < 0.05; **, P < 0.01).

Figure 3

PUM regulation of candidate gene protein levels. A, RT-PCR from triple-negative breast cancer cells MDA-MB-231 for PTEN, NRAS, c-JUN, and FOXO1. B, Western blots from siRNA-treated MDA-MB-231 cells. C, 3′ RACE RT-PCR from CAL51 cells for c-JUN. D, Western blots of CAL51 cells transfected with individual siRNAs targeting GFP, PUM1, and PUM2. E, Cell number assay from CAL51 cells transfected with individual siRNAs. F, Number of HRAS-mediated invasive cells from wild-type (WT) or c-JUN-null MEFs (JUN^−/−), infected with the c-JUN coding sequences and either 3′ UTR isoform (*, P < 0.05).

Figure 4

The effect of APA on mRNA stability and protein production on candidate genes. A, Table, change in PRE number (Δ in PRE) and total PRE after APA of FOXO1. Graph, luciferase assays comparing FOXO1 full-length and extended 3′ UTR isoforms in MDA-MB-231 cells depleted of PUM1, PUM2, or control Scrambled sequence (Scr; **, P < 0.01). Full-length 3′ UTR isoform set to 1. B, mRNA stability assays of the FOXO1 full-length or extended 3′ UTR isoforms from MDA-MB-231 cells treated with the polymerase inhibitor, actinomycin D (**, P < 0.01; *, P < 0.05). C, Table, transcript information for the shortened 3′ UTR of PTEN. Graph, luciferase assays comparing PTEN full-length and short 3′ UTR isoforms in MDA-MB-231 cells depleted of PUM1, PUM2, or Scrambled (**, P < 0.01). D, mRNA stability assays of the PTEN short or long 3′ UTR isoforms from MDA-MB-231 cells treated with actinomycin D (**, P < 0.01). E, Table, transcript information for the shortened 3′ UTR of NRAS. Graph, luciferase assays comparing NRAS full-length and short 3′ UTR isoforms in MDA-MB-231 cells depleted of PUM1, PUM2, or Scrambled (*, P < 0.05). F, mRNA stability assays of the NRAS short or long 3′ UTR isoforms from MDA-MB-231 cells treated with actinomycin D. G, Table, transcript information for the shortened 3′ UTR of c-JUN. Graph, luciferase assays comparing c-JUN full-length and short 3′ UTR isoforms in MDA-MB-231 cells depleted of PUM1, PUM2, or Scrambled (*, P < 0.05; **, P < 0.01). H, mRNA stability assays of the c-JUN short or long 3′ UTR isoforms from MDA-MB-231 cells treated with actinomycin D (**, P < 0.01).

Figure 5

APA in triple-negative breast tumors correlates with slower growth, smaller yet more invasive tumors. A, Tumor size of triple-negative breast tumors, divided by grade and APA status (*, P < 0.05). Grade 2 tumors (n = 6), grade 3 APA tumors (n = 12), and grade 3 non-APA tumors (n = 12). B, Ki67 levels of triple-negative breast tumors, divided by grade and APA status (*, P < 0.05). C, Lymph node status (positive or negative) from patients with triple-negative breast tumors, divided by grade and APA status. D, RT-PCR analysis of PUM1 and PUM2 from triple-negative breast tumors, divided by grade and APA status. E, 3′ RACE experiments from MDA-MB-231 cells depleted of PUM1, PUM2, PABPN1, or Scrambled control sequence by shRNA. Change in 3′ UTR length calculated by dividing distal values with proximal readings. F, RT-PCR analysis of the polyadenylation machinery from triple-negative breast tumors, divided by grade and APA status (**, P < 0.01). G, RT-PCR measuring mRNA length from cells transfected with either the pCDNA-empty vector or pCDNA-CSTF3 (**, P < 0.01; *, P < 0.05).

Figure 6

Triple-negative breast cancer cell lines are more dependent on CSTF3. A, Expression of CSTF3 from triple-negative breast cancer cell lines (TNBC) versus breast cancer cells from other breast cancer subtypes. B, CSTF3 expression levels from SK-BR-3 (non-TNBC), BT-20 (TNBC), and BT-549 (TNBC) cells. C, Relative CSTF3 depletion from the three cell lines using independent siRNAs. D, Cell numbers from SK-BR-3, BT-20, and BT-549 depleted of GFP or CSTF3 by siRNA (**, P < 0.01; *, P < 0.05).

Figure 7

The polyadenylation machinery utilizes GU-rich sequences of the PUM complex to catalyze APA. A, Schematic of the positioning and sequence composition of motifs recognized by the CFI, CSTF, and PUM complexes. B, Model describing how the CFI and CSTF complex can use motifs of the PUM complex for APA.