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. Author manuscript; available in PMC: 2015 Jun 16.
Published in final edited form as: Cancer Cell. 2014 May 15;25(6):748–761. doi: 10.1016/j.ccr.2014.04.008

Sumoylation Pathway Is Required to Maintain the Basal Breast Cancer Subtype

Maria V Bogachek 1, Yizhen Chen 1, Mikhail V Kulak 1, George W Woodfield 1, Anthony R Cyr 1, Jung Min Park 2, Philip M Spanheimer 1, Yingyue Li 1, Tiandao Li 1,3, Ronald J Weigel 1,2,4
PMCID: PMC4096794  NIHMSID: NIHMS587621  PMID: 24835590

SUMMARY

The TFAP2C/AP-2γ transcription factor regulates luminal breast cancer genes and loss of TFAP2C induces epithelial-mesenchymal transition. By contrast, the highly homologous family member, TFAP2A, lacks transcriptional activity at luminal gene promoters. A detailed structure-function analysis identified that sumoylation of TFAP2A blocks its ability to induce the expression of luminal genes. Disruption of the sumoylation pathway by knockdown of sumoylation enzymes, mutation of the SUMO-target lysine of TFAP2A, or treatment with sumoylation inhibitors induced a basal to luminal transition, which was dependent upon TFAP2A. Sumoylation inhibitors cleared the CD44+/hi/CD24−/low cell population characterizing basal cancers and inhibited tumor outgrowth of basal cancer xenografts. These findings establish a critical role for sumoylation in regulating the transcriptional mechanisms that maintain the basal cancer phenotype.

INTRODUCTION

Breast cancer has an incidence of 226,000 and accounts for approximately 40,000 deaths annually in the US (Siegel et al., 2012). There has been an improvement in survival for women with breast cancer, though patients with locally advanced or metastatic disease continue to have a poor prognosis. The clinical subtypes of breast cancer are defined by the expression of estrogen receptor-alpha (ERα), progesterone receptor (PgR) and amplification and overexpression of c-ErbB2/HER2. The four common molecular subtypes of breast cancers include the Luminal A (ERα/PgR+, HER2−), Luminal B (ERα/PgR+, HER2+), HER2 (ERα/PgR−, Her2+) and triple-negative (ERα/PgR−, HER2−) (Carey et al., 2006; Sorlie et al., 2001). The luminal breast cancer subtypes (comprising approximately 75% of breast cancer in postmenopausal women) are characterized by the expression of a set of ERα-associated genes (Sorlie et al., 2001). Although it is well established that patterns of gene expression in breast cancer are predictive of clinical phenotype, little is known about the transcriptional mechanisms responsible for establishing the characteristic expression profile. Since many of the ERα-associated genes are not part of the ERα pathway, the co-expression of these genes suggests the existence of transcriptional mechanisms common to luminal genes.

The triple-negative breast cancer subtype is a heterogeneous group that represents 10–20% of breast cancers (Bertucci et al., 2012; Lehmann et al., 2011). The triple-negative subtypes have an aggressive clinical course and do not respond to therapy effective for cancers that express ERα or HER2. Hence, there has been intense research focus on understanding the molecular characterization of this group with the goal of defining novel molecular targets (Bertucci et al., 2012). Detailed molecular profiling has allowed further subclassification of the triple-negative breast cancer phenotypes into at least six distinct subtypes including basal-like 1, basal-like 2, immunomodulatory, mesenchymal-like, mesenchymal stem-like and luminal androgen receptor subtypes (Lehmann et al., 2011). Other proposed sub-classifications of the triplenegative breast cancer phenotype have identified a claudin-low subgroup characterized by the relatively reduced expression of genes involved in cell adhesion and formation of tight junctions (Herschkowitz et al., 2007; Valentin et al., 2012). Basal-like breast cancers are further distinguished from luminal cancers by frequent mutations of TP53, gene expression patterns characteristic of epithelial-to-mesenchymal transition (EMT) and an increase in the percentage of cancer stem cells (CSC) (Bertucci et al., 2012; Valentin et al., 2012).

TFAP2C (AP-2γ) is a member of the developmentally regulated family of AP-2 factors that include five members—TFAP2A (AP-2α), TFAP2B (AP-2β), TFAP2C (AP-2γ), TFAP2D (AP-2δ) and TFAP2E (AP-2ε) (Bosher et al., 1996; Feng and Williams, 2003; Moser et al., 1995; Williams et al., 1988; Zhao et al., 2001). TFAP2C binds to a GC-rich consensus sequence in the promoters of target genes through a helix-loop-helix motif in the DNA binding domain (Eckert et al., 2005). Analysis of a ChIP-seq data set for TFAP2C defined the consensus site as the nine base sequence SCCTSRGGS (S = G/C, r = A/G) (Woodfield et al., 2010), which closely matches the previously defined optimal in vitro binding site (McPherson and Weigel, 1999). AP-2 factors are expressed early in differentiation of the ectoderm and specify cell fates within the epidermis and neural crest (Hoffman et al., 2007; Li and Cornell, 2007). Within the adult mammary gland, TFAP2C is expressed in the luminal and myoepithelial cells (Cyr et al., 2014; Friedrichs et al., 2005; Friedrichs et al., 2007). Overexpression of TFAP2A or TFAP2C in mouse mammary epithelial cells (MMEC) results in lactation failure with hypoplasia of the alveolar mammary epithelium during pregnancy (Jager et al., 2003; Zhang et al., 2003). Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in MMEC promoted aberrant growth of the mammary tree leading to a reduction in the luminal cell population and concomitant gain of the basal cell population at maturity (Cyr et al., 2014). In tumor models, both TFAP2A and TFAP2C are important to cell proliferation, establishment of colonies in soft agar, cell migration and xenograft outgrowth (Orso et al., 2008).

In breast cancer, AP-2 factors regulate expression of both ERα and Her2. TFAP2C regulates expression of ERα as well as other ERα-associated genes characteristic of luminal breast cancer (Cyr et al., 2014; deConinck et al., 1995; McPherson et al., 1997; Woodfield et al., 2007). TFAP2A and TFAP2C induce expression of the cloned HER2/ErbB2 promoter (Begon et al., 2005; Bosher et al., 1996; Delacroix et al., 2005; Yang et al., 2006). TFAP2C bound to the HER2 promoter and knockdown of TFAP2C reduced HER2 expression (Ailan et al., 2009). In BT474 breast carcinoma cells, TFAP2A and TFAP2C coordinately regulate HER2 expression (Allouche et al., 2008) and a correlation has been established between AP-2 expression and the expression of HER2 in primary breast cancers (Allouche et al., 2008; Pellikainen et al., 2004; Turner et al., 1998).

Several critical questions remain to be addressed. There is 83% similarity between TFAP2A and TFAP2C with 76% identity in the carboxyl-half of the proteins containing the DNA binding and dimerization domains (McPherson et al., 1997). In neural crest development, TFAP2A and TFAP2C appear to have complementary and overlapping roles (Hoffman et al., 2007). However, in breast cancer models, TFAP2C was found to have a unique role in regulation of ESR1/ERα gene expression, which was functionally distinct from the effects of TFAP2A (Woodfield et al., 2007). Furthermore, recent findings have highlighted a critical role for TFAP2C in maintaining the luminal phenotype through the induction of luminal-associated genes and repression of basal-associated genes (Cyr et al., 2014). It remains to be seen if TFAP2A has a similar effect on the expression of luminal genes. Furthermore, if the role of TFAP2C were functionally distinct, it would be of critical importance to understand the molecular basis for transcriptional specificity of luminal gene regulation. We expect that mechanisms regulating patterns of gene expression in breast cancer would provide important insight into strategies for drug development. With these considerations in mind, we sought to confirm the functional differences between TFAP2A and TFAP2C in regulation of luminal gene expression and to determine the molecular basis for functional specificity of TFAP2C-mediated gene regulation.

RESULTS

Functional Specificity of TFAP2C for the Luminal Gene Expression Cluster

Previous studies in luminal breast cancer cell lines demonstrated that knockdown of TFAP2C down-regulated ERα, whereas, knockdown of TFAP2A failed to have a similar effect on ERα expression (Cyr et al., 2014; Woodfield et al., 2007). In order to validate the unique functional role of TFAP2C in primary human cancer, we obtained fresh tumor tissue from patients with ERα-positive breast cancer. Tumor-derived breast cancer cells were transduced with lentiviruses encoding TFAP2A, TFAP2C or non-targeting shRNA. As seen in Figures 1A–C and S1A, knockdown of TFAP2C but not TFAP2A repressed expression of ERα, confirming that TFAP2C has unique functional effects with regard to ESR1/ERα gene regulation and that the cell line models are reflective of gene regulation in primary human breast cancer. Using MCF-7 cells, a more expansive examination of luminal gene targets was performed. The luminal breast cancer subtype expresses a set of luminal-associated genes including ESR1/ERα, MUC1, FGFR4, KRT8, RET, MYB, FOXA1 and GATA-3 (Kao et al., 2009). Knockdown of TFAP2C repressed expression of luminal genes, as noted by analysis of RNA (Figure 1D, S1B) and protein (Figure 1E), whereas, knockdown of TFAP2A had minimal or no effect. The basal target genes, MMP14, CALD1 and CD44, which are overexpressed in basal cancers, were repressed by TFAP2C but not TFAP2A. By contrast, a known TFAP2A target gene, CDKN1A/p21-CIP (Scibetta et al., 2010; Woodfield et al., 2007), was responsive to TFAP2A only (Figure 1D, E). Hence, although TFAP2A has the ability to induce certain genes, it lacks functional activity with regard to the luminal-associated gene expression cluster.

Figure 1. Functional Specificity of TFAP2C for the Luminal Cluster Genes.

Figure 1

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A. Primary ERα-positive breast cancer cells derived from patient samples were transduced with lentiviral vectors encoding shRNA specific for non-targeting (NT), TFAP2A (A) or TFAP2C (C). Knockdown of TFAP2A and TFAP2C was confirmed compared to NT (data for tumor 2 and 3 shown in Figure S1A), *p<0.05 compared to NT. B. ERα RNA was assessed by RT-PCR; data for all three tumor isolates demonstrates that knockdown of TFAP2C specifically repressed ERα expression, * p<0.05 compared to NT. C. Western blot for ERα protein confirmed that ERα protein expression was repressed by knockdown of TFAP2C only. D. Functional effects on RNA expression of luminal genes, the basal genes, MMP14, CALD1 and CD44, and the TFAP2A-specific target gene CDKN1A/p21-CIP in MCF-7 cells after knockdown of either TFAP2A or TFAP2C; data demonstrates functional specificity of TFAP2C (additional luminal genes in Figure S1) with statistical differences shown comparing knockdown of TFAP2A vs. TFAP2C for all genes (p<0.05) and * p<0.05 compared to NT control. E. Western blot confirmed functional specificity for TFAP2C in regulation of luminal and basal genes. Error bars indicate SEM. See also Figure S1.

One possibility for the functional differences of TFAP2A and TFAP2C might be due to differences in the ability for the factors to bind to the regulatory regions of the luminal cluster genes. In addition to the genes examined above, the FREM2 gene was identified as a specific TFAP2C target gene, which was highly responsive to changes in TFAP2C expression but was unresponsive to TFAP2A (Figure S1). Using ChIP-seq the binding of TFAP2A and TFAP2C was compared in MCF-7 cells (Figure 2A). An analysis of the genomic binding of TFAP2A and TFAP2C to the regulatory regions of the luminal-associated genes, ESR1/ERα, FOXA1 and FREM2, demonstrated co-localization of the two factors. ChIP-seq data for the other luminal target genes examined demonstrated identical binding patterns for TFAP2A and TFAP2C in the promoter regulatory regions and these data agreed with other published ChIP-seq data for MCF-7 cells (Figure S2). Epitope-tagged AP-2 constructs were used to confirm the identical chromatin binding patterns (Figure 2B–C). HA-tagged constructs for TFAP2A and TFAP2C were transfected into MCF-7 cells and ChIP was performed with anti-HA antibody with amplification at on-target and off-target sites for ESR1/ERα, FOXA1 and FREM2 (Woodfield et al., 2010). The data demonstrate that TFAP2A and TFAP2C bind to the AP-2 sites in the luminal target genes with approximately equal binding affinity. Hence, the functional differences between TFAP2A and TFAP2C cannot be attributed to differences in genomic binding.

Figure 2. ChIP of TFAP2A and TFAP2C with Functional Specificity of TFAP2C Mapped to Amino Terminus.

Figure 2

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A. ChIP-Seq demonstrates identical binding pattern comparing TFAP2A and TFAP2C to luminal target genes ESR1/ERα, FOXA1 and FREM2; red dot indicates peak analyzed in detail in part C. B. Western blot of MCF-7 cells transfected with empty vector (EV) or HA epitope tagged AP-2 constructs, TFAP2C (HA-C) or TFAP2A (HA-A), and probed with antibody shown. C. Real-time ChIP was performed with anti-HA antibody and precipitated chromatin amplified at off-target and on-target locations for ESR1/ERα, FOXA1 and FREM2 (Woodfield et al., 2010). Data confirm specific binding of TFAP2A and TFAP2C to peaks identified by ChIP-seq with minimal binding to off-peak sites. D. Schematic of TFAP2A (blue) and TFAP2C (yellow) showing homologous regions and chimeric AP-2 proteins generated (all chimeras generated using TFAP2C-mut, which is construct insensitive to the siRNA); AD: Activation Domain; Di: Dimerization Domain; DBD: DNA Binding Domain; assignment of functional domains described previously (Williams and Tjian, 1991a; Williams and Tjian, 1991b). E. Using endogenous ERα RNA expression as functional assay, MCF-7 cells were transfected with siRNA and expression vector as diagramed in D. The data show that rescue of ERα transcriptional activity maps to the amino half of the TFAP2C protein. F. Experiment identical to part E, except uses expression of endogenous FREM2 and maps functional effect to the first 128 amino acids of TFAP2C. *p<0.05 compared to normalized expression in untransfected control. Error bars indicate SEM. See also Figure S2.

Functional Specificity of TFAP2C Localized to Amino Terminus

We sought to identify the domain of TFAP2C responsible for regulation of the luminal cluster genes. A knock-down/knock-in system was developed in which the expression of endogenous TFAP2C was knocked-down by siRNA and rescued by co-transfection with expression vectors for either TFAP2A or TFAP2C, engineered to be resistant to the siRNA. Chimeric AP-2 proteins were created where regions of TFAP2A were substituted with the homologous region of TFAP2C (Figure 2D). Using endogenous ERα expression as a marker for activation, rescue of ERα expression was localized to the amino-half of TFAP2C (Figure 2E). FREM2 was more robust as a marker for TFAP2C-specific activation and allowed localization of luminal-specific activation to the first 128 amino acids of the activation domain (Figure 2F).

Sumoylation Pathway Inhibits Transcriptional Activity of TFAP2A

The luminal cluster gene promoters appear to share a common transcriptional mechanism and we sought to understand the molecular basis for the functional block of TFAP2A. The block of TFAP2A activity at luminal promoters might involve either co-activator(s) specific for TFAP2C or promoter-specific co-repressors of TFAP2A. A set of potential AP-2 co-factors were identified using yeast two-hybrid in which either TFAP2C or TFAP2A was used as bait (Figure 3A). A set of factors was chosen based on previous findings suggesting a potential role in gene regulation. The functional effect of each cofactor was assessed by serial knockdown using specific siRNAs and assaying for expression of endogenous FREM2 (Figure 3B). Of 21 AP-2 binding partners tested, knockdown of two TFAP2A-interactive factors, PIAS1 and Ubc9/UBE2I significantly induced endogenous FREM2 expression in MCF-7 cells. Ubc9 is a unique E2 SUMO-conjugating enzyme (Ihara et al., 2008; Johnson and Blobel, 1997) and PIAS1 is a SUMO E3 ligase (Leitao et al., 2011). Ubc9 was previously shown to bind to TFAP2A and TFAP2C (Eloranta and Hurst, 2002). As both proteins are part of the sumoylation pathway, the findings implicated sumoylation as a potential mechanism accounting for a functional block of TFAP2A in regulation of the luminal gene cluster. GST pull-down and co-immunoprecipitation confirmed that Ubc9 bound to both TFAP2A and TFAP2C (Figure 4 A–C). Knockdown of Ubc9 increased endogenous FREM2 expression (Figure 4D). Whereas, knockdown of TFAP2A alone had no effect, knockdown of TFAP2A abrogated the effect of Ubc9 knockdown, indicating that in the absence of the sumoylation pathway, FREM2 expression became responsive to TFAP2A. Interestingly, Western blot of TFAP2A often demonstrates a doublet (Figures S1G, 4D and previous publications (Woodfield et al., 2010)). Knockdown of Ubc9 reduced the relative amount of the upper band (Figure 4D), suggesting the potential for sumoylation of TFAP2A. One major site for sumoylation is lysine 10 and the TFAP2A isoform 1A, which was used to generate the chimeric AP-2 proteins, contains the K10 SUMO site (Berlato et al., 2011). To formally demonstrate sumoylation of TFAP2A, SUMO-1, -2 and -3 were expressed in vitro with TFAP2A. Wild-type TFAP2A was sumoylated by all three SUMO proteins but the TFAP2A mutant K10R had significantly reduced sumoylation (Figure 4E). In MCF-7 cells, wild-type TFAP2A but not K10R mutant was sumoylated in vivo with all three SUMO proteins (Figure 4F). Immunoprecipitated TFAP2A was evaluated by western blot with anti-SUMO2/3 antibody and demonstrated the endogenous sumoylated form of TFAP2A in MCF-7 cells (Figure 4G). Although sumoylated forms of TFAP2C were identified in vitro with SUMO-1, -2 or -3 (Figure 4H), expression in MCF-7 cells in vivo was only able to identify sumoylation of TFAP2C with co-expression of SUMO-1 (Figure 4I). The sumoylated forms of AP-2 were estimated to be 70 ± 5 kDa (Figure S3A–D). Sumoylation of TFAP2A was similarly demonstrated in the basal line BT549, which was increased with peroxide (Figure S3E, F).

Figure 3. Yeast Two-hybrid Identifies Sumoylation Pathway Regulating Activity of TFAP2A on FREM2.

Figure 3

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A. Yeast two-hybrid screen using TFAP2A or TFAP2C as bait identified potential AP-2 interacting factors. Proteins from named genes are shown that were uniquely pulled out using either TFAP2A (blue) or TFAP2C (yellow) or were pulled out with both factors (green) as bait. B. Set of 21 factors was chosen for screening by knockdown with specific siRNAs in MCF-7 cells and assaying for effects on expression of endogenous FREM2 compared to NT siRNA (normalized to 1.0), *p<0.05 compared to NT. Two proteins identified as TFAP2A-interacting factors in yeast two-hybrid screen (PIAS1 and Ubc9/UBE2I) significantly induced FREM2 expression with knockdown. Error bars indicate SEM.

Figure 4. Sumoylation Functionally Linked to AP-2 Activity.

Figure 4

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A. Ubc9 binds to TFAP2A and TFAP2C in GST pull-down. B. MCF-7 cells were transfected with expression vectors for Green Fluorescent Protein-AP-2 fusion proteins demonstrating nuclear expression of GFP fusion proteins with co-localization with DAPI nuclear stain. C. Co-IP of GFP-TFAP2A and GFP-TFAP2C confirms protein-protein interaction between Ubc9 and both AP-2 proteins. D. Expression of endogenous FREM2 RNA in MCF-7 cells transfected with siRNA (normalized to NT) indicated with western blots below. Data show that FREM2 expression is not responsive to TFAP2A; however, knockdown of Ubc9 induced FREM2 expression and induction is blocked with knockdown of TFAP2A showing that FREM2 expression responds to TFAP2A in absence of sumoylation pathway, p<0.05 compared to NT. Error bars indicate SEM. E. Sumoylation using in vitro assay demonstrates wild-type TFAP2A is sumoylated by SUMO-1, -2 or -3, whereas, TFAP2A K10R mutant has significantly reduced sumoylation. F. MCF-7 cells co-transfected with expression vector for TFAP2A or K10R mutant construct for SUMO-1, -2 or -3. Data show that wild-type TFAP2A is sumoylated in vivo, whereas the K10R mutant is not. G. Protein from MCF-7 cells was immunoprecipitated (IP) using IgG or anti-TFAP2A antibody; protein was eluted from beads (E) and prewashes (P1 and P2) were assayed; load is unprecipitated extract; western blot was probed with anti-SUMO2/3 antibody. H. TFAP2C was sumoylated in vitro with SUMO-1, -2 or -3; *indicates sumoylated form of TFAP2C. I. MCF-7 cells transfected with expression vectors for SUMO-1, -2 or -3 and western blot probed with TFAP2C shows evidence for sumoylation with SUMO-1; *indicates location of sumoylated TFAP2C. See also Figure S3.

Inhibiting SUMO Conjugation of TFAP2A Induced a Basal to Luminal Transition

To demonstrate the functional effects of sumoylation, wild-type and the K10R mutant of TFAP2A were transfected into MCF-7 cells. Transfection of TFAP2A-K10R induced expression of luminal-associated genes (RET, MUC1 and FREM2), whereas, transfection of wild-type TFAP2A had no effect (Figure 5A). By contrast, transfection of wild-type TFAP2A or K10R mutant induced expression of CDKN1A/p21-CIP. The induction of FREM2 protein with expression of TFAP2A-K10R was confirmed by Western blot, without changes in expression of TFAP2C (Figure 5B). Though the effects on luminal gene expression were reproducible, the overall effects were modest since parental MCF-7 cells express TFAP2C as well as the luminal gene targets. We have shown that stable knockdown of TFAP2C converts MCF-7 cells from a luminal to basal-like phenotype (Cyr et al., 2014). Hence, a MCF-7 cell clone with stable knockdown of TFAP2C (sKD-C) was utilized compared to a cell clone with a nontargeting shRNA (sKD-NT). Overexpression of TFAP2A-K10R in sKD-C basal-like cells significantly induced the luminal genes ESR1/ERα, KRT8 and FREM2, whereas wild-type TFAP2A had no effect (Figure 5C). Inhibiting sumoylation by knockdown of either PIAS1 or Ubc9 in sKD-C cells resulted in re-activation of ERα mRNA and protein expression and repressed expression of the basalassociated gene CD44 (Figure 5D). To confirm the effect of the sumoylation pathway, the small molecule inhibitor of sumoylation, ginkgolic acid (GA) (Fukuda et al., 2009), was examined for its effect on ERα, FREM2 and CD44 expression. Treatment of sKD-C cells with GA induced ERα and FREM2 expression and repressed CD44 expression compared to vehicle treatment without altering TFAP2C expression (Figure 5E).

Figure 5. Functional Effects of TFAP2A-K10R Mutant and Sumoylation Inhibition.

Figure 5

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A. Expression of luminal genes in MCF-7 cells transfected with wildtype TFAP2A or K10R mutant. K10R mutant induced luminal genes but wild-type TFAP2A did not. Both wild-type and K10R mutant TFAP2A induced CDKN1A/p21. B. Protein expression from western blots performed in triplicate with example of western blot below showing FREM2 protein expression induced by K10R but not wild-type TFAP2A. C. Expression of luminal cluster genes ESR1/ERα, KRT8 and FREM2 in sKD-C cells transfected with empty vector (EV) or expression vector for TFAP2A or K10R-TFAP2A mutant, with expression normalized to EV. K10R induced expression of luminal target genes, whereas, wild-type TFAP2A did not. D. Knockdown of Ubc9 or PIAS1 re-activated ERα and repressed CD44 expression in sKD-C cells. E. Treatment of sKD-C cells with ginkgolic acid (GA) re-activated FREM2 and ERα and repressed CD44 mRNA normalized to lowest value (top) and protein (bottom). For all panels, * indicates p<0.05 compared to normalized signal of 1.0. For CD44 expression in panel E, GA treated and untreated were also significantly different from each other, p<0.05. Error bars indicate SEM.

Sumoylation Inhibitors Clear the CD44+/hi/CD24−/low Cell Population

The findings suggest that the sumoylation pathway plays a critical role in maintaining the basal breast cancer phenotype. One of the characteristics of basal breast cancers is the relatively high percentage of the CD44+/hi/CD24−/low cell population. To confirm the generality of the findings, basal breast cancer cell lines were treated with GA or another inhibitor of sumoylation, anacardic acid (AA) (Fukuda et al., 2009). As previously reported, we confirmed that AA inhibited SUMO conjugation of proteins globally and the SUMO conjugated form of TFAP2A specifically (Figure S4). AA was also noted to decrease slightly the overall expression of TFAP2A. As seen in Figure 1, knockdown of TFAP2C induced a 1.5-fold increase in TFAP2A, indicating that TFAP2C moderately represses TFAP2A. The finding that AA induced in a slight decrease in TFAP2A is consistent with the SUMO un-conjugated form of TFAP2A acquiring TFAP2Clike repression activity. Treatment of the basal breast cancer cell lines BT-20 and BT-549 as well as sKD-C cells with sumoylation inhibitors abrogated expression of CD44 and significantly reduced the CD44+/hi/CD24−/low population (Figure 6A). In addition, cells from a primary basal breast cancer were treated in vitro in parallel. The cancer was from a patient with locally advanced breast cancer that was refractory to conventional chemotherapy. Remarkably, treatment with GA or AA cleared the CD44+/hi/CD24−/low population from the cells harvested from the primary tumor (Figure 6A, last panel). By contrast, GA/AA treatment of MCF-10A cells, a normal breast cell line model, had no effect on CD44 expression.

Figure 6. Sumoylation Inhibitors Cleared CD44+/hi/CD24−/low Cell Population.

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A. Treatment of s-KD-C, BT-549, BT-20 or cells derived from a primary basal cancer (Basal Cancer) with GA or anacardic acid (AA) inhibited CD44 expression by Western blot (top row) and significantly reduced the CD44+/hi/CD24−/low population by FACS analysis (lower panels) but had no effect on the normal breast cell line MCF-10A. B/C. Western blots showing that CD44 repression by GA and AA treatment was dependent upon expression of TFAP2A since knockdown of TFAP2A with siRNA abrogated effect of sumoylation inhibitors in sKD-C cells (B) and basal cell lines BT549 and BT20 (C). See also Figure S4.

To further demonstrate the role of AP-2 in repression of CD44, the effects of GA and AA were examined with knockdown of TFAP2A. As seen in Figures 6B and 6C, knockdown of TFAP2A alone had no effect on CD44 expression in sKD-C, BT549 and BT20 cells. However, the ability for GA and AA to repress CD44 expression in the basal breast cancer cell lines was completely eliminated by knockdown of TFAP2A. Since drug effects might not be specific for a single pathway, GA and AA might induce changes in CD44 expression through several mechanisms. To prove that the effects on CD44 expression were mediated through the sumoylation pathway, Ubc9 and PIAS1 were knocked down by siRNA in the basal cell lines BT549 and BT20 and the effects on CD44 expression were assessed. As seen in Figures 7A and 7B, knockdown of either Ubc9 or PIAS1 similarly repressed CD44 expression. Furthermore, knockdown of Ubc9 or PIAS1 eliminated the SUMO conjugated form of TFAP2A (and slightly reduced the overall level of TFAP2A) in both basal (BT549) and luminal (MCF-7) cells (Figures 7C and 7D).

Figure 7. Knockdown of Sumoylation Enzymes Repressed CD44 and Blocked SUMO Conjugation of TFAP2A.

Figure 7

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Knockdown of Ubc9 and PIAS1 by siRNA repressed expression of CD44 in BT549 (A) and BT20 (B) cells showing same effect as GA and AA. Error bars indicate SEM. C/D. Endogenous TFAP2A was examined by western blot in BT549 (basal) (C) and MCF-7 (luminal) (D) cells. The SUMO-conjugated form of TFAP2A is seen in both cell types (denoted by *) and knockdown of either Ubc9 or PIAS1 significantly reduced SUMOconjugated TFAP2A. MW shows molecular weight markers. E. On left, tumor-free survival of nude mice (n=5 per group) inoculated with BT20 cells pretreated for 48 hours with either GA or AA compared to no pre-treatment; pretreated cells failed to form tumors. On right, tumor-free survival of nude mice (n=5 per group) inoculated with BT20 cells with animals gavaged with AA vs. vehicle. F. On left, IOWA-1T cells were pre-treated with anacardic acid (AA) or vehicle and mice were followed until requiring euthanasia due to tumor size (Overall Survival). Pre-treatment with AA inhibited tumor formation, n=5 animals per group, p=0.002. On right, nude mice were inoculated with 1×106, 5×105, or 2.5×105 IOWA-1T cells and animals were gavaged with either vehicle or AA; vehiclegavaged mice developed tumors in 5, 10 and 12 days, respectively. Mice gavaged with AA failed to form tumors over the course of the experiment, n=3 animals per group, p=0.025.

The CD44+/hi/CD24−/low cell population is associated with a subset of breast carcinoma cells that are critical for the initiation of tumor xenografts. To address the effects of GA and AA on tumor initiation, BT20 cells were pretreated with GA or AA compared to vehicle prior to inoculation of xenografts in nude mice. As noted in Figure 7E, pretreatment of the cells repressed the formation of tumor xenografts, whereas, vehicle treated cells formed xenografts with a median time of 8 weeks. To prove that the drugs were not cytotoxic, BT20 xenografts were inoculated in nude mice and the animals were gavaged with AA or vehicle. As noted in Figure 7E, animals gavaged with AA failed to form tumors, whereas, vehicle gavaged animals formed xenografts as expected. We created a breast carcinoma cell line (IOWA-1T) from the basal tumor-derived cells used in Figure 6A. The IOWA-1T cell line rapidly forms locally advanced tumors in nude mice (unpublished data). Identical experiments using the IOWA-1T basal cell line confirmed that either pre-treating the cancer cells or gavaging animals with AA repressed tumor initiation of xenografts (Figure 7F).

DISCUSSION

Transcriptional Regulation of Luminal Gene Expression in Breast Cancer

In the presence of an intact sumoylation pathway, TFAP2C transcriptionally regulates the expression of luminal genes such as ERα/ESR1, whereas, TFAP2A is functionally inactive at luminal gene promoters. A model depicting the role of AP-2 factors in establishing gene expression patterning in breast cancer is presented in Figure 8. Further, the data herein support the conclusion that this model is reflective of gene regulation in primary ERα-positive cancer. Recent evidence indicates that TFAP2C likely functions in concert with other transcription factors, including ERα and FOXA1, to regulate many of the genes in the luminal expression cluster (Cyr et al., 2014; Jozwik and Carroll, 2012; Tan et al., 2011). Other studies have attempted to define mechanisms common to the regulation of luminal breast cancer genes (Joshi et al., 2012). GATA-3 regulates the differentiated luminal breast cancer phenotype (Fang et al., 2009) and recent findings indicate an important role of FOXM1 in mediating mammary luminal cell differentiation through GATA-3 (Carr et al., 2012). Previous studies have also implicated FOXA1 (Bernardo et al., 2013), Elf5 (Chakrabarti et al., 2012), BRCA1 (Bai et al., 2013) and ErbB3 (Balko et al., 2012) in maintaining the luminal mammary phenotype. Interestingly, prostate-derived ETS factor (PDEF) mediates luminal differentiation and also correlates with expression in luminal breast cancer suggesting a strong link between development of luminal mammary cells and oncogenesis of luminal breast cancer (Buchwalter et al., 2013). Similarly, TFAP2C participates in luminal mammary development and luminal gene expression in breast cancer (Cyr et al., 2014), further strengthening the link between the processes of luminal differentiation and oncogenesis.

Figure 8. Schematic Model of Gene Regulation in Breast Cancer Subtypes.

Figure 8

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In scenario 1 with an intact SUMO pathway and active TFAP2C, breast cancer cells are maintained in the luminal phenotype. Transcriptional regulation of many luminal gene promoters are coordinately regulated by TFAP2C, ERα and FOXA1, as well as potentially other co-factors. As diagrammed in scenario 2, loss of TFAP2C in the presence of the SUMO pathway induces epithelialmesenchymal transition (EMT), characterized by repression of luminal genes and induction of basal-associated genes. As shown in scenario 3, inhibition of the SUMO pathway in the absence of TFAP2C activity, SUMO unconjugated TFAP2A induces mesenchymal-epithelial transition (MET), characterized by repression of basal-associated gene expression and induction of luminalassociated genes.

Sumoylation Blocked the Activity of TFAP2A at Luminal Gene Promoters

Several lines of evidence indicate that sumoylation plays a key role in establishing the functional differences between TFAP2C and TFAP2A and accounts for the functional block of TFAP2A at luminal gene promoters (see Figure 8). First, we have demonstrated sumoylation of TFAP2A at lysine 10 in vitro and in vivo. Second, blocking the sumoylation pathway either by knockdown of critical enzymes in the sumoylation pathway or with the use of small molecule inhibitors of sumoylation allowed TFAP2A to induce expression of luminal genes such as ESR1/ERα and FREM2 and repress expression of the basal gene CD44. Finally, mutation of the SUMO target lysine of TFAP2A conferred the ability to induce expression of luminal cluster genes. Taken together the data indicate that sumoylation of endogenous TFAP2A blocks this factor from regulating luminal gene expression. Furthermore, it is clear that the functional effect of sumoylation is a luminal gene-specific effect since wild-type or K10R mutant TFAP2A was active in transcriptional activation of CDKN1A/p21-CIP. Although there is evidence that TFAP2C can be sumoylated, we were not able to demonstrate that sumoylation has functional effects on the transcriptional activity of TFAP2C. Under conditions where sumoylation of TFAP2A blocked its activity at luminal gene promoters, TFAP2C remained active despite evidence for similar levels of sumoylation. Further studies concerning details of sumoylation of TFAP2C may uncover subtle effects at specific promoters.

The Role of Sumoylation in Cancer-Related Gene Regulation

Sumoylation involves the post-translational modification of proteins through the covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues in target proteins (Bettermann et al., 2012; Cubenas-Potts and Matunis, 2013). At least four SUMO proteins have been described, SUMO-1-4. The enzymatic pathway involves several steps beginning with ATP-dependent activation of the SUMO protein by the E1 heterodimer ASO1-UBA2, continuing with the transfer of SUMO to the cysteine residue of the E2 enzyme Ubc9, and finally the enzymatic transfer of the SUMO tag to the target protein by the E3 ligase, e.g. PIAS1. Sumoylation of key regulatory proteins influences several aspects of oncogenesis and cancer progression (Bettermann et al., 2012). There is a growing body of literature reporting the sumoylation of transcription factors and the effects on transcriptional regulation (Gill, 2003) including sumoylation of androgen receptor (AR), glucocorticoid receptor (GR), C/EBP, Smad4, Myb, Ets-1, Pdx1, Sp3, p300, CREB and p53 (Bettermann et al., 2012). In most cases, sumoylation represses transcriptional activity. Mechanisms resulting in transcriptional repression by sumoylation may include effects of protein stability, altered cellular localization or DNA binding, modulation of co-repressor binding and altered association with chromatin modifying enzymes such as histone deacetylases (HDACs) (Gill, 2003; Girdwood et al., 2003). Holmstrom et al. (Holmstrom et al., 2008) showed that transcriptional inhibition by sumoylation occurred at compound but not single sites and was related to the ability for sumoylation to destabilize the transcription factor-chromatin interaction. A mechanism whereby sumoylation destabilizes TFAP2A binding to certain regulatory regions may provide a mechanism for our finding of promoter-specific repression. Since the function of TFAP2A at promoters for genes such as CDKN1A/p21-CIP is SUMO-insensitive, it is possible that promoter regulatory structure common to luminal genes may account for SUMO-specific effects. Many luminal genes contain closely linked promoter elements for AP-2, ERα and FOXA1 (Tan et al., 2011), and the interaction of these factors may be sensitive to sumoylation (Figure 8). Consistent with previous studies, sumoylation of TFAP2A was increased by peroxide and confirms that oxidative stress can increase sumoylation of factors (Bossis and Melchior, 2006; Ryu et al., 2010). Recent studies have suggested that the sentrin-specific protease 1 (SENP1) may be involved in SUMO de-conjugation of factors in breast cancer (Abdel-Hafiz and Horwitz, 2012; Chen et al., 2013). It is intriguing to consider that SENP1 may regulate transcriptional activity of AP-2 factors in breast cancer by inducing SUMO de-conjugation. Further work is needed to elucidate details of the transcriptional mechanisms whereby sumoylation specifically inhibits the functional activity of certain factors such as TFAP2A.

Sumoylation Inhibitors Clear the CD44+/hi/CD24−/low Cell Compartment

Basal breast cancers are characterized by a relatively high percentage of cells expressing the characteristic markers CD44+/hi/CD24−/low, which include tumor-initiating cells associated with the outgrowth of tumor xenografts (Al-Hajj et al., 2003; Iqbal et al., 2013). The CD44+/hi/CD24−/low population is relatively chemo-resistant and becomes enriched after chemotherapy (Lee et al., 2011). Stable knockdown of TFAP2C in luminal cancer cells induced epithelial-mesenchymal transition (EMT), characterized by the repression of luminal gene expression, activation of basal-associated genes and an increased the population of cells expressing the CD44+/hi/CD24−/low markers (Figure 8) (Cyr et al., 2014). In the current study, SUMO inhibition allowed TFAP2A to acquire TFAP2C-like repression activity, inhibiting CD44 expression, clearing cells expressing CD44+/hi/CD24−/low markers and blocking the outgrowth of cancer xenografts. Of particular clinical relevance, sumoylation inhibitors were able to efficiently clear the CD44+/hi/CD24−/low population in a primary basal breast cancer obtained from a patient with a locally advanced breast cancer that was refractory to conventional chemotherapy. The high percentage of cells expressing the CD44+/hi/CD24−/low markers was likely due to selection from the treatment with chemotherapy. The remarkable effect of SUMO inhibitors to clear the CD44+/hi/CD24−/low tumor-initiating cell population suggests that this class of agents may have an effect on basal breast cancers either alone or in combination with conventional chemotherapy. Since the CD44+/hi/CD24−/low population defines the tumor initiating cells in many types of carcinomas, it is possible that SUMO inhibitors may have clinical effects in a wide range of carcinomas. Interestingly, sumoylation inhibitors did not affect MCF10A cells, which are commonly used as a model for normal breast cells. Hence, the sumoylation pathway appears to be critical for maintaining the basal breast cancer subtype and is not a general mechanism regulating CD44 expression in normal breast cells.

EXPERIMENTAL PROCEDURES

Cell lines

The human breast cancer cell lines were derived and maintained as described (Cyr et al., 2014). Under a protocol approved by the University of Iowa IRB and with informed consent, primary cancer cells were obtained from surgical resection specimens and cell suspensions were prepared with gentle collagenase/hyaluronidase (Stemcells) (Ponti et al., 2005). The IOWA-1T cell line was established from a primary basal tumor (unpublished data).

Chromatin Immunoprecipitation with Direct Sequencing (ChIP-Seq)

ChIP-Seq was performed as described (Woodfield et al., 2010).

Sumoylation Assays

Sumoylation in vitro was done using SUMOlink™ SUMO-1 Kit and SUMOlink™ SUMO-2/3 Kit (Carlsbad). SUMO plasmids and pcDNA3.1-TFAP2A or K10R mutant were used for in vitro protein production. MCF-7 were transfected for 48 hours with 2 µg SUMO expressing plasmids (Feng et al., 2013) that were kindly provided by Dr Xiaolu Yang (University of Pennsylvania). As indicated in some experiments, the proteasome inhibitor MG132 was added as described (Chu and Yang, 2011).

Western blots

Western blots were performed as previously described (Cyr et al., 2014; Kulak et al., 2013).

AP-2 Constructs

AP-2 constructs were amplified using previously cloned cDNAs for template (McPherson and Weigel, 1999). Gateway TFAP2A and TFAP2C clones were inserted in-frame into pG-LAP1(Torres et al., 2009) via LR clonase reaction using Gateway LR Clonase II Enzyme Mix (Invitrogen).

Gingolic and Anacardic Acid Treatment

Cells were plated (2.5×105/10cm2) and treated with 10 µM gingolic or anacardic acid (Sigma) for 2–4 days and collected for qPCR, Western blot and FACS analysis.

Flow cytometry

FACS analysis was performed as previously described (Cyr et al., 2014; Roederer and Hardy, 2001) (http://www.flowjo.com/v8/html/distancing.html).

Yeast two-hybrid assay

Yeast two-hybrid for AP-2 factors was performed as previously described (McPherson et al., 2002).

Tumor Xenografts

Following a vertebrate animal protocol approved by the University of Iowa IACUC, xenografts were generated by inoculating 5×106 BT20 cells or indicated number of IOWA-1T cells into nude mice as previously described (Woodfield et al., 2007). All experiments conformed to the regulatory standards reviewed in the animal protocol.

Statistical Analysis

Statistical analysis was performed using the two-sided Student’s T-test for continuous variables. Comparisons for xenografts were performed using the logrank test.

Accession number

The ChIP-seq data are accessible in GEO database under accession number GSE44257.

Supplementary Material

01

Highlights.

  • Maintenance of the luminal or basal breast cancer phenotype is a dynamic process

  • TFAP2C has a unique role in maintaining the luminal breast cancer phenotype

  • SUMO inhibition causes TFAP2A to acquire ability to regulate luminal genes

  • SUMO inhibition clears the CD44+/hi/CD24−/low population in basal breast cancers

SIGNIFICANCE.

Clinical breast cancer subtypes are characterized by patterns of gene expression that predict outcome and response to therapy. Luminal breast cancers express steroid hormone receptors and tend to be hormone responsive. By comparison, basal breast cancers are not hormone responsive, display an expansion of a CD44+/hi/CD24−/low cell population and have a worse prognosis. Herein, we show that sumoylation of the TFAP2A transcription factor is required to maintain the basal breast cancer phenotype. Disruption of SUMO conjugation of TFAP2A was associated with a loss of the CD44+/hi/CD24−/low cell population and an associated inability for basal cancer lines to form tumor xenografts. Inhibiting the sumoylation pathway may be an effective treatment strategy for basal breast cancer.

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health grants R01CA109294 (PI: R.J. Weigel), T32CA148062 (PI: R. J. Weigel) and by a generous gift from the Kristen Olewine Milke Breast Cancer Research Fund. PMS was supported by the NIH grant T32CA148062. This research was supported in part through computational resources provided by The University of Iowa, Iowa City, IA.

Footnotes

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