Arginine methyltransferases as novel therapeutic targets for breast cancer

Abstract

Breast cancer is the most commonly diagnosed female cancer in the world. Though therapeutic treatments are available to treat breast cancer and in some instances are successful, the occurrence of unsuccessful treatment, or the rate of tumour recurrence, still remains strikingly high. Therefore, novel therapeutic treatment targets need to be discovered and tested. The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyse arginine methylation and are implicated in a myriad of cellular pathways including transcription, DNA repair, RNA metabolism, signal transduction, protein–protein interactions and subcellular localisation. In breast cancer, the expression levels and enzymatic activity of a number of PRMTs is dysregulated; significantly altering the regulation of many cellular pathways that are implicated in breast cancer development and progression. Here, we review the current knowledge on PRMTs in breast cancer and provide a rationale for how PRMTs may provide novel therapeutic targets for the treatment of breast cancer.

Introduction

Worldwide, breast cancer is the most common cancer observed in women accounting for 25% of all cancers, with 1.7 million new cases diagnosed in 2012 (1). Like all cancers, breast cancer arises through the dysregulation of cellular pathways regulating sensitivity to growth stimuli, cellular proliferation, replicative potential, apoptosis, angiogenesis and tissue invasiveness and metastasis (2). Though therapeutic options are available for the treatment of breast cancer including surgical resection, radiation and chemotherapy, in many instances tumours become refractory to these treatments or the tumours may recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease.

Arginine methylation, a common post-translation modification catalysed by a family of enzymes called the protein arginine methyltransferases (PRMTs) is implicated in a number of cellular processes including transcription, DNA repair, RNA metabolism, signal transduction, protein–protein interactions and subcellular localisation (3). PRMTs catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the guanidine nitrogen of an arginine residue, resulting in the formation of a methylarginine and S-adenosylhomocysteine (AdoHcy) as a by-product (3). The addition of methyl groups to a substrate promotes increased bulkiness and local hydrophobicity which can affect protein–protein interactions, albeit without changing the cationic charge of the arginine residue (4).

Members of the PRMT family are characterised by four conserved sequence motifs (I, post-I, II and III), and ‘double E’ and ‘THW’ loops (5, 6). There are currently nine identified human PRMTs (PRMT 1–9) in mammals. PRMTs are classified based on the type of arginine methylation that they catalyse. Both type I and type II PRMTs produce monomethylarginines as an intermediate, with type I PRMTs (PRMT1, 2, 3, 4(CARM1), 6, 8) catalyzing the formation of asymmetric dimethylarginines (ADMA), while type II PRMTs (PRMT5) form symmetric dimethylarginines (SDMA). Type III PRMTs (PRMT7) are capable of forming stable monomethylarginine (MMA) products (7), while the enzymatic activity of PRMT9 has yet to be fully characterised (8) (Figure 1). Most PRMTs methylate arginine residues within a glycine- and arginine-rich (GAR) motif (9). However, CARM1and PRMT7 display unique substrate specificity, as CARM1 methylates arginine residues within a proline-, glycine- and methionine-rich (PGM) motif (10), and PRMT7 methylates arginine residues in a RXR motif (11). PRMT5 can methylate arginine residues in both a GAR and PGM motif (12, 13), while PRMT6 is efficient at methylating arginine residues both within the GAR motif, in addition to arginine residues independent of this sequence (14–17).

PRMTs and breast cancer

As PRMTs operate in a plethora of central cell regulatory pathways, it would be hypothesised that dysregulation of their expression might have adverse effects on cellular and tissue homeostasis. Indeed, a growing body of evidence has linked PRMTs to the development and progression of cancer. Aberrant expression of PRMTs has been characterised in human tumours including, lung, colorectal, bladder, prostate, leukaemia and breast (18–24). PRMTs 1–7 have been linked with breast cancer development and progression and this review will outline the current knowledge regarding the proposed role of these PRMTs in breast cancer and elaborate on their potential as novel therapeutic targets for breast cancer.

PRMT1

PRMT1 is the main arginine methyltransferase in cells, accounting for ~85% of all arginine methylation activity (25, 26). Complete depletion of PRMT1 expression results in a loss of cell viability and embryonic lethality in mice (27, 28). Functionally, PRMT1 has been implicated in transcription activation, signal transduction, RNA splicing and DNA repair (29–33). Alternative splicing of the 5′ end of the PRMT1 pre-mRNA yields seven distinct isoforms (v1–v7) with each isoform having a unique N-terminal sequence and a slightly different molecular weight. Each PRMT1 isoform has distinct enzymatic properties, substrate specificity and subcellular localisation, and thus it would be hypothesised that they should be functionally distinct (34).

In breast cancer tumour samples, PRMT1 mRNA expression is upregulated compared to adjacent normal tissue with PRMT1 expression correlating with patient age, menopausal status, tumour grade and status of the progesterone receptor (22, 35). Examination of the expression of PRMT1v1, v2 and v3 in breast cancer samples identified a strong correlation between PRMT1v1 mRNA expression and poor patient prognosis whereas PRMT1v2 and PRMT1v3 expression did not correlate with any clinical or pathological indicators at least at the RNA level (22). Immunohistochemical examination of total PRMT1 protein expression, not differentiating between specific isoforms, identified increased PRMT1 expression within the cytoplasm (22). Since PRMT1v2 localises predominantly to the cytoplasm, while PRMT1v1 displays nuclear localisation (34, 36), it is interesting to speculate that the observed increased cytoplasmic expression of PRMT1 may actually represent increased PRMT1v2 expression. In a panel of breast cancer cell lines, increased mRNA and protein expression of all PRMT1 isoforms was observed relative to normal mammary epithelial cells, though expression of PRMT1v2 was increased to an extent greater than the other isoforms. Additionally, increased PRMT1v1 and v2 mRNA expression was observed in breast tumour tissue compared to normal tissue (34). Interestingly, contradictory results were obtained pertaining to PRMT1v2 mRNA expression as Mathioudaki et al. (22) saw no increase in PRMT1v2 expression in breast tumour samples, while Goulet et al. (34) describe increased expression in breast cancer cell lines and in a tissue sample. Further assessment of PRMT1v2 mRNA expression in a greater number of breast tumour samples from different types and disease stages will be required to address this potential discrepancy.

PRMT1v2 is the only PRMT1 isoform containing exon 2 within its coding sequence. Exon 2 encodes a CRM1-dependent nuclear export sequence which distinguishes PRMT1v2 as the only PRMT1 isoform with predominantly cytoplasmic localisation. Examination of the contribution of PRMT1v2 to breast cancer indicated an involvement for PRMT1v2 in promoting cell survival and cellular invasion (37). Specific depletion of PRMT1v2, without affecting the expression of PRMT1v1, the predominant PRMT1 isoform resulted in apoptotic cell death. Furthermore, PRMT1v2 depletion caused a significant decrease in cell motility and invasion in highly aggressive MDA MB 231 breast cancer cells; a decrease significantly greater than what could be accounted for from the observed apoptosis. Conversely, overexpression of PRMT1v2 increased motility and invasion in mildly aggressive MCF7 cells; a phenotype dependent on PRMT1v2’s cytoplasmic localisation and methyltransferase activity. While overexpression of PRMT1v1 was also able to enhance cell motility in MCF7 cells, augmented invasion was a phenotype specific to PRMT1v2 overexpression. PRMT1v2 overexpression in MCF7 cells resulted in disruption of adherens junctions, with dissociation of F-actin, E-cadherin and β-catenin from these junctions (37). Disruption of adherens junction stability is associated with increased invasive potential and metastasis in numerous epithelial-derived cancers, including breast cancer (38–43). PRMT1v2-mediated cell invasion occurs through phosphorylation-dependent downregulation of β-catenin (37). β-catenin protein stability is regulated through phosphorylation of serine residues 33 and 37 and threonine residue 41 by a degradation complex composed of axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β), which negatively regulates WNT signalling through the degradation of β-catenin (44–46). Cha et al. have shown that PRMT1 functions to negatively regulate β-catenin protein expression through the methylation of axin. Both PRMT1v1 and PRMT1v2 were capable of methylating axin, in vitro. Axin methylation by PRMT1 results in increased axin protein stability; therefore enabling an enhanced interaction with GSK3β, thus promoting β-catenin degradation. Conversely, PRMT1 depletion results in increased cytoplasmic β-catenin and β-catenin-dependent transcriptional activity (47).

Additionally, PRMT1 influences signalling pathways aberrantly regulated in breast cancer pathology. Presence of the estrogen receptor (ER) in a breast tumour is an important prognostic and predictive biomarker (48). Upon rapid estrogen stimulation, PRMT1 methylates estrogen receptor α (ERα) on arginine residue 260, resulting in the assembly of a complex containing ERα, the p85 subunit of phosphoinositide 3-kinase (PI3K), Src and focal adhesion kinase (FAK). Assembly of this complex results in an activating phosphorylation on serine 473 on Akt, a downstream target of this pathway whose activation promotes cellular proliferation and anti-apoptotic signalling. Mutation of arginine residue 260 or PRMT1 depletion attenuates ERα-mediated activation of Akt. Moreover, in breast cancer patient samples, an increase in ERα methylation is observed, in comparison to adjacent normal mammary epithelium (49–51). Alterations in gene expression of several nodes of the PI3K/Akt signalling pathway are frequent in ER positive breast cancer (52). In accordance with results observed by Le Romancer et al. (49), Simoncini et al. (53) show that estrogen stimulation of ERα promotes receptor binding to the p85α subunit of PI3K resulting in downstream activation of Akt. Thus, it appears that the therapeutic targeting of PRMT1 may be beneficial in attenuating ER-mediated activation of the PI3K/Akt signalling pathway. Furthermore, methylated ERα is present exclusively in the cytoplasm (49); therefore, it could be hypothesised that PRMT1v2 is the isoform responsible for depositing this methyl mark as it is the only isoform which displays predominant cytoplasmic localisation (34, 36). Further experimentation will be required to determine if this is true.

Interestingly, a number of studies have illustrated conflicting roles for PRMT1 in regulating apoptosis. PRMT1 methylation of forkhead box O1 (FOXO1) at arginine residues 248 and 250 attenuates Akt phosphorylation of FOXO1 at serine 253. Inhibition of Akt phosphorylation facilitates nuclear localisation of FOXO1 which promotes oxidative-stress induced apoptosis (54). In accordance with the above study, in MCF7 cells, PRMT1 methylates the pro-apoptotic protein, BCL-2 antagonist of cell death (BAD) promoting apoptosis. PRMT1 methylation of BAD prevents its phosphorylation by Akt, which would result in BAD’s mitochondrial sequestration and binding to the anti-apoptotic protein BCL-X_L, and thus inhibit apoptosis (55). However, in contrast to these two studies, an inhibitory methylation mark by PRMT1 on the apoptotic signal-regulating kinase 1 (ASK1) prevents its activation, protecting cells from stress induced apoptosis in MCF7 and MDA MB 231 cells (56). Although discrepancies have arisen regarding PRMT1’s function in regulating apoptosis, these discrepancies could potentially be due to PRMT1 isoform specific functions in apoptosis, as the above studies focused on the role of PRMT1 in general. Further experimentation will be required to delineate if besides PRMT1v2 other PRMT1 isoforms function in apoptotic or cell survival pathways, and if their functions are contradictory or redundant.

PRMT1 null mouse embryonic fibroblasts (MEFs) exhibit spontaneous DNA damage and aberrant cell cycle regulation leading to chromosomal irregularities that eventually lead to cell death (57). Therefore, it is not surprising that PRMT1 functions in pathways responsible for maintaining genomic stability through the regulation of specific target proteins. PRMT1 methylates breast cancer 1, early onset (BRCA1), a tumour suppressor protein involved in DNA damage pathways. Genetic mutation of the BRCA1 gene results in increased predisposition to the development of breast cancer (58). BRCA1 was shown to be methylated in both breast cancer cell lines and patient breast tumour samples. PRMT1 methylation of BRCA1 occurs within the DNA-binding region, mediating not only BRCA1’s ability to bind its target genes but also its ability to interact with other proteins (59).

PRMT1 regulates additional components of the DNA damage pathway thereby regulating their function. PRMT1 methylation of the DNA damage sensing protein p53-binding protein 1 (53BP1) regulates its recruitment to sites of DNA double-strand breaks (32). Furthermore, PRMT1 methylates MRE11, a component of the MRE11/RAD50/NBS1 (MRN) complex responsible for repairing DNA double-stranded breaks, mediating its cellular localisation and exonuclease activity (60, 61). Additionally, PRMT1 has also been implicated in maintaining telomere length. PRMT1 methylates TRF2, a component of the shelterin complex which functions to control telomere length and stability, with depletion of PRMT1 in cancer cells resulting in telomere shortening (62). Finally, upon ultraviolet irradiation, PRMT1 functions synergistically with CARM1 and the acetyltransferase p300 as transcriptional co-activators of p53, regulating its target gene expression (63). These studies exhibit that PRMT1 exudes an integral role in the maintenance of genomic stability.

The transforming growth factor β (TGFβ)/Smad signalling pathway has been implicated in breast cancer through stimulating the de-differentiation of epithelial cells to malignant fibroblastic cells capable of invasion and metastasis (64, 65). PRMT1 was recently identified as a component of the TGFβ signalling pathway in response to bone morphogenetic protein (BMP) binding to TGFβ receptors, RI and RII (66). Activation of the TGFβ receptor occurs through ligand binding and dimerization of the RI and RII receptors (67). Smad6 typically sequesters the TGFβ RI receptor holding it in a dormant state, with PRMT1 binding the TGFβ RII receptor. Upon stimulation of the TGFβ receptor though BMP ligand binding, PRMT1 methylates Smad6 resulting in dissociation of Smad6 from the RII receptor, promoting RI and RII dimerization and activation of the receptor. TGFβ receptor activation through BMP stimulation has been shown to promote cancer cell invasion (68).

Since PRMT1 accounts for the majority of the identified methyltransferase activity in the cell (25, 26), it is not surprising that abnormal regulation of PRMT1 has been associated with cancer development and progression in mammary epithelial cells. PRMT1 mRNA and protein expression are increased in breast cancer tumours (22, 35), and PRMT1 was shown to contribute to pathways promoting increased cellular proliferation, invasion and survival in breast cancer cell lines (35, 37, 49, 56, 66). The majority of the published data pertaining to PRMT1 has focused on the contribution of total PRMT1 to breast cancer, not differentiating between the contributions of specific isoforms. It was recently shown that PRMT1v2 promotes cell survival, migration and invasion while PRMT1v1 stimulates cell migration in breast cancer cells (37). Therefore, future work should focus on the contribution of the specific isoforms in breast cancer and elucidating each of their roles in breast cancer, as all were shown to be upregulated in breast cancer cell lines (34).

Similarly, future work should additionally focus on how PRMT1 expression and its enzymatic activity are regulated. PRMT1 interacts with the mammalian-early gene TIS21/BTG2/PC3, the leukaemia-associated B-cell translocation gene (BTG1) (69), and CCR4-associated factor 1 (hCAF1) (70), with both BTG2 (71) and hCAF1 (70) capable of modulating PRMT1 methyltransferase activity in a substrate-dependent manner. Furthermore, hCAF1 was found to interact with both BTG1 and BTG2 to mediate transcription of ERα target genes (72). BTG1 and BTG2 typically function to inhibit cell proliferation and both have been implicated in the development of breast cancer. Decreased BTG1 expression correlates with poor patient prognosis in breast cancer patient samples (73, 74), while BTG2 maps to chromosome 1q32, a region frequently altered in breast adenocarcinomas (75, 76). Therefore, it is plausible that under normal growth conditions BTG1, BTG2 and hCAF1 control PRMT1 enzymatic activity, and in breast cancer cells, loss of BTG1 and/or BTG2 removes this regulation on PRMT1 activity allowing PRMT1 to function promiscuously. Further investigation will be required to prove if this hypothesis is true. The published data suggests that dysregulation of PRMT1 contributes significantly to breast oncogenesis, and although further experimentation will elaborate on PRMT1’s role in breast cancer, investigating PRMT1 as a novel therapeutic target in breast cancer may prove beneficial.

PRMT2

PRMT2 was identified through its sequence homology (~50%) with the catalytic domain of PRMT1 (77). Unlike PRMT1 null mice which display embryonic lethality, PRMT2 null mice are viable; although PRMT2 null MEFs exhibit increased NF-κB transcriptional activity and are more resistant to apoptosis than wild-type MEFs (78,79). PRMT2 exhibits weak methyltransferase activity relative to PRMT1 (80). With the exception of arginine residue 8 on histone 3, identified substrates for PRMT2 are lacking (81). However, PRMT2 contains an N-terminal Src homology 3 (SH3) binding domain, which has identified functions in cell proliferation, migration and cytoskeletal modifications (82). Therefore, this does not exclude the possibility that additional yet unidentified PRMT2 substrates exist, or in some cellular processes, PRMT2 may function independent of its catalytic activity.

PRMT2 undergoes alternative splicing to produce five distinct isoforms: full length PRMT2 and four truncated isoforms PRMT2L2, PRMT2α, β, and γ that occur through alternative splicing in exons 7–10 of the mRNA. PRMT2L2, PRMT2α, β and γ have lost conserved motifs III and the THW loop which forms part of the AdoMet binding pocket, and thus may exhibit no methyltransferase activity (83, 84). Expression of each isoform (PRMT2, PRMT2L2, PRMT2α, β and γ) was investigated in breast cancer cell lines, and it was discovered that PRMT2 mRNA and protein expression of each isoform is increased in a panel of ER positive breast cancer cell lines in comparison to a panel of ER negative breast cancer cell lines. In breast tumour samples, PRMT2 mRNA expression of all of the isoforms is increased; consistent with breast cancer cell lines, PRMT2 expression in ER+ tumours is increased relative to ER− tumours (83, 84). Furthermore, PRMT2 protein expression, not differentiating between isoforms, was also elevated in breast tumours with a similar increase in expression observed in ER+ tumours relative to ER− tumours (84).

PRMT2 interacts with and enhances the activity of the estrogen and progesterone receptors (PR) in a ligand-dependent manner (85). Binding of the ERα receptor by all PRMT2 isoforms was shown to enhance estrogen-mediated transactivation of the receptor augmenting promoter activity of the transcription factor Snail (83, 84). Increased Snail expression in cancer cells is consistent with enhanced cellular invasion (86). In contrast to PRMT1 which methylates ERα (49), to date methylation of the ER by PRMT2 has not been documented. Intriguingly, in response to estrogen stimulation, PRMT2 negatively regulates cell proliferation and colony formation in ER+ breast cancer cells, while PRMT2 depletion increases tumour growth in a MCF7 xenograft mouse model (84, 87). PRMT2-mediated growth suppression in ER+ breast cancer cell lines was discovered to occur through modulation of the E2F/cyclin D1 pathways. Depletion of all PRMT2 splice variants coincides with an enhancement in E2F expression and activity (84). This observation is consistent with PRMT2 binding the retinoblastoma (Rb) protein causing E2F repression (78). PRMT2 regulates cyclin D1 by indirectly binding to the AP-1 site on the cyclin D1 promoter, and through suppression of the Akt/GSK-3β signalling pathways (87). PRMT2 displays predominantly nuclear localisation (83, 84). Loss of PRMT2 nuclear expression in breast tumour samples correlates with increased cyclin D1 expression, thus promoting breast tumour cell proliferation (87).

These results suggest that in contrast to PRMT1 which promotes breast tumour development and progression, PRMT2, at least in ER+ breast tumours suppresses cell proliferation, with loss of nuclear PRMT2 expression a potential driving force to tumour development. Interestingly, in ER+ breast cancer cell lines, PRMT2L2, a predominantly cytoplasmic variant of PRMT2 displays equal or increased mRNA expression compared to full length PRMT2 which localises predominantly to the nucleus (83). This suggests that abnormal alternative splicing of PRMT2 could at least in part be one mechanism that can facilitate ER+ breast tumour development. PRMT2 alternatively spliced variants PRMT2L2, PRMT2α, β and γ have lost conserved motifs III and the THW loop which form part of the AdoMet-binding motif, and thus should be enzymatically inactive (83, 84). Therefore, these variants may function to promote breast tumorigenesis through co-transactivation of the ER or other unknown transcription factors, or through other functions potentially mediated through PRMT2’s N-terminal SH3 binding domain.

PRMT3

PRMT3 shares 67% sequence similarity with PRMT1 in the catalytic methyltransferase domain. PRMT3 distinguishes itself from other members of the PRMT family as it contains a zinc-finger domain at its N-terminus, which is thought to function as a substrate recognition module (88). PRMT3 null mice are viable. Although mutant embryos are small, the mice attain normal size in adulthood (89). Though, to date a limited number of PRMT3 substrates have been identified, PRMT3 does methylate the ribosomal protein RPS2 to regulate ribosome homeostasis (89, 90), as well as p53 in an ARF and VHL30 dependent manner (91).

Though no studies have alluded to aberrant expression of PRMT3 in breast cancer, the tumour suppressor protein DAL-1/4.1B, whose expression is frequently lost in breast tumour samples (92, 93), binds to PRMT3, negatively regulating its methyltransferase activity (94). These results suggest that breast tumours may exhibit higher PRMT3 methyltransferase activity in tumours where loss of DAL-1/4.1B is observed. Further experimentation will be required to determine if PRMT3 expression and/or enzymatic activity is altered in breast tumours and if loss of DAL-1/4.1B binding to PRMT3 were to mediate any potential effects.

CARM1 (PRMT4)

PRMT4, best known as co-activator associated methyltransferase 1 (CARM1) exhibits a unique substrate specificity, where it methylates arginine residues within a proline-, glycine- and methionine-rich (PGM) motif instead of a GAR motif (10, 12). CARM1 null mice survive through gestation, though they are small and die shortly after birth; a phenotype mimicked in transgenic mice expressing catalytically inactive CARM1. CARM1 null embryos display differentiation defects in T cells, adipose tissue, chondrocytes, muscles and lungs (95–102). CARM1 functions in a myriad of cellular processes including transcription regulation (103), mRNA splicing (12, 104), cell cycle progression (105) and the DNA damage response (106, 107), through methylation of identified substrates including histones (108, 109), transcription factors (12, 110), co-activators (111–114), splicing factors (12, 109) and RNA polymerase II (115).

A number of studies have described a role for CARM1 in breast cancer aetiology and progression. CARM1 acts as a co-activator for ERα-mediated transcription in a ligand-independent manner. This association with ERα is dependent on phosphorylation by cAMP-associated protein kinase A at CARM1 serine residue 448 and CARM1 auto-methylation at arginine residue 551 (116, 117). In addition to a ligand-independent interaction with ERα, CARM1 is necessary for estrogen-stimulated proliferation of breast cancer cells which occurs through CARM1 methylation of H3R17, resulting in expression of the cell cycle regulator E2F1. Upon estrogen stimulation, CARM1 also regulates the protein stability and promotes the activity of the transcription factor AIB1, a protein frequently overexpressed in breast tumours (118). It has been observed that CARM1 functions synergistically with the proto-oncogene proline-, glutamic acid- and leucine-rich protein 1 (PELP1) to enhance ERα activity through PELP1 modulation of CARM1’s H3 arginine methylation at the promoters of ERα target genes (119). PELP1 promotes breast cancer invasion and metastasis, and functions as a co-activator of ERα. In breast tumours, PELP1 expression correlates with poor patient prognosis (120–124). Pharmacologic inhibition or depletion of CARM1 in ER+ breast cancer cells inhibits PELP1-mediated breast cancer cell proliferation, migration and anchorage-independent growth (119). Furthermore, CARM1 expression correlated with PELP1 expression in ER+ tumours, and was elevated in metastatic breast tumours compared to normal breast tissue (119). This data suggests that CARM1 functions synergistically with at least PELP1 to promote tumourigenesis in ER+ breast cancers.

Additional studies have further corroborated this detected increase in CARM1 expression in breast cancer samples. CARM1 expression was observed to be upregulated in breast tumours with CARM1 expression correlating with tumour size and grade, as well as Ki-67, TK1, CD71 and cyclin E expression (122, 123). Consistent with CARM1 expression correlating with cyclin E expression, CARM1 was shown to be recruited to the promoter of cyclin E1 where it functions as a transcriptional co-activator of cyclin E1 expression. This regulation coincided with a correlative increase in CARM1 and cyclin E1 expression in grade 3 breast tumours (105). Furthermore, CARM1 expression is increased in non-luminal breast tumours displaying HER2 expression (125, 126). Davis et al. (127) further elaborate on CARM1 expression in breast cancer through classification of CARM1 cellular localisation in correlation to distinct histo-pathological and molecular tumour subtypes, as well as expression of prognostic markers. The authors reasoned that since CARM1 functions as a transcription regulator in steroid pathways (118, 128), the majority of CARM1 in a breast cancer cells should be localised within the nucleus. However, an equal distribution of nuclear and cytoplasmic CARM1 was observed in ductal carcinoma in situ, and invasive and metastatic histological subtypes; although, in basal-like and HER2+ breast tumours a significant increase in cytoplasmic CARM1 localisation occurred (127). Interestingly, no correlation was detected between both nuclear or cytoplasmic CARM1 and the ER, as CARM1 was previously shown to interact and stimulate the activity of the ER in a ligand-dependent and independent manner (116, 118). No correlation was discerned between the PR and CARM1, although CARM1 cytoplasmic localisation did correlate with expression of both the HER2 and EGFR receptors. Lastly, cytoplasmic CARM1 expression was found to correlate with poor patient prognosis (127). Altogether, these results suggest that in addition to functioning as a co-activator of ERα, CARM1 may perform additional functions in promoting tumorigenesis in HER2+ and basal-like tumours that require CARM1 cytoplasmic expression.

In contrast to these studies, Al-Dhaheri et al. show CARM1 overexpression in ER+ breast cancer cells inhibiting estrogen-stimulated cell proliferation through repression of estrogen-activated target genes. Depletion of CARM1 in MCF7 cells resulted in an increase in tumour growth in a xenograft mouse model. CARM1 overexpression and the diminution in estrogen-stimulated cell proliferation coincided with an increase in expression of the cyclin-dependent kinase inhibitors p21 and p27, and a change in cell morphology towards a more differentiated phenotype. Lastly, CARM1 protein expression correlated with ERα expression with higher CARM1 expression detected in lower grade ER+ breast tumours (129).

CARM1 also methylates arginine residue 1064 of BAF155, the core subunit of the SWI/SNF chromatin remodeling complex. Methylated BAF155 expression was increased in breast patient samples, with methylated BAF155 promoting colony formation and cell migration, in vitro, and metastasis in an in vivo lung metastatic mouse model (130).

Though contradictory data has arisen for the role of CARM1 in breast cancer, the majority of data suggests that CARM1 functions to promote breast cancer development and progression. CARM1 is required for the ligand dependent and independent activation of ERα promoting transcription of ERα target genes (116, 118); however whether CARM1 expression is changed in ER+ breast tumours will require further investigation. CARM1 cellular localisation was previously observed to be cell type dependent (131). This could explain why increased cytoplasmic CARM1 expression was detected in only HER2+ and basal-like breast tumours (127) and not in other subtypes of breast cancer due to dysregulation of CARM1 expression in these particular tumours. CARM1 appears to promote breast cancer regardless of the specific subtype; although the molecular mechanisms regulated by CARM1 in the development and/or progression of breast cancer have yet to be completely elucidated. Nevertheless, the published data suggests that CARM1 may represent a novel therapeutic target.

PRMT5

PRMT5 is, thus far, the only characterised symmetric methyltransferase. Complete loss of PRMT5 results in loss of cell viability and mouse embryonic lethality, with PRMT5 required for embryonic epiblast cell differentiation (132). PRMT5 participates in a wide variety of cellular processes including transcription repression (133), alternative splicing (134–136), signal transduction (137–140), ribosome biogenesis (141), assembly of the Golgi apparatus (142), cellular differentiation (102, 143, 144), and germ cell specification (145, 146).

In breast cancer cells, PRMT5 methylates programmed cell death 4 (PDCD4) on arginine residue 110 (147). PDCD4 has been characterised as a tumour suppressor protein with lower expression observed in invasive breast carcinoma compared to normal mammary epithelium (148). In addition, PDCD4 expression is capable of suppressing anchorage-independent cell growth (149). Surprisingly, xenografts in severe combined immunodeficiency (SCID) mice of MCF7 cells co-overexpressing PDCD4 and PRMT5 display enhanced tumour growth which is dependent on PRMT5 methyltransferase activity. PRMT5 expression in breast cancer patient samples was observed to vary between tumour samples, though high expression of both PDCD4 and PRMT5 correlates with poor patient outcome (147).

Like PRMT3, DAL-1/4.1B can regulate PRMT5 methyltransferase activity (150). However, in contrast to PRMT3 where repression of PRMT3 methyltransferase activity was observed, DAL-1/4.1B modulates PRMT5 methyltransferase activity in a substrate-dependent manner. Interestingly, Jiang et al. (151) show DAL-1/4.1B co-operates with protein methylation to induce caspase 8-dependent apoptosis in MCF7 cells. Though, in this study, methylation was inhibited with adenosine-2′-3′-dialdehyde (Adox), a general methylation inhibitor, it is intriguing to speculate based on DAL-1/4.1B modulation of PRMT3 and PRMT5 activity that DAL-1/4.1B collaborates with one or both of these arginine methyltransferases to induce apoptosis. Furthermore, in breast cancer, could DAL-1/4.1B loss coincide with altered PRMT3 and/or PRMT5 enzymatic activity possibly correlating with a propensity for these enzymes to promote oncogenesis. Subsequently, does DAL-1/4.1B influence PRMT5 enzymatic activity towards PDCD4? Further investigations will be required to discover if this is the case.

PRMT6

PRMT6, a predominantly nuclear protein was identified as the first PRMT possessing the capacity for auto-methylation (14). Subsequently, it has been shown that this auto-methylation which occurs on arginine residue 35 is necessary to maintain PRMT6 protein stability (152). PRMT6 shares a high sequence similarity with the other PRMTs within the main AdoMet-catalytic domain. Similar to PRMT1, PRMT6 contains only the core PRMT catalytic domain with no additional domains present within its sequence (14). PRMT6 null mice are viable, although PRMT6 null MEFs undergo rapid senescence (153). PRMT6 displays distinct substrate specificity from other PRMTs and primarily functions as a transcriptional regulator through methylation of H3R2 (153–157), H3R42 (158) and H2AR29 (159).

Contradictory evidence has arisen pertaining to PRMT6 expression levels in breast cancer. Yoshimatsu et al. (35) identified increased PRMT6 expression in about 33% of breast cancer patient samples with increased PRMT6 expression effectuating poor patient prognosis. In support of this study, Phalke et al. (157) show increased PRMT6 expression in both breast cancer cell lines and patient tumour samples. However, Dowhan et al. (160) demonstrate that PRMT6 expression was significantly down-regulated in invasive ductal carcinomas. Therefore, further research will be required to clarify the precise impact PRMT6 expression exacts on breast tumours with a more thorough examination of PRMT6 expression in different histo-pathological and molecular subsets of breast cancer providing a more concise view as to its contribution.

PRMT6 functions in numerous cellular pathways including transcription regulation, alternative splicing, DNA repair and cell cycle regulation, all of which have been linked to breast cancer aetiology. PRMT6 transcriptionally represses the anti-angiogenic factor thrombospondin 1 (TSP-1) concurring with an increase in cell migration and invasion (155); however, overexpression of PRMT6 was also shown to induce TSP-1 expression, coinciding with a decrease in cellular migration and invasion due to downregulation of MMP2 and MMP9 (161). Furthermore, PRMT6 methylation of H3R2 promotes the transcriptional repression of HoxA10 (154), a protein whose upregulation in breast cancer promotes increased p53 expression and reduced invasive potential (162). Interestingly, PRMT6 transcriptionally represses the expression of TERT, Golph3 and prothymosin α through H3R2 methylation, all genes implicated in promoting breast cancer (154). Additionally, H2AR29 methylation represses MMP9 transcription (159); a matrix metalloproteinase involved in breast cancer pathology (163–168). Contrary to its function as a transcriptional repressor, PRMT6 can also function as a transcription activator by mediating the transcriptional activity of the PR, as well as working synergistically with CARM1 to promote expression of the ER (169).

PRMT6 methylates the high mobility group A1a protein (HMGA1a) within its second AT hook domain on arginine residues 57 and 59; a region critical for protein-DNA and protein–protein interactions (15, 170, 171). HMGA1a belongs to the high mobility group family of proteins which function as master switches of the chromatin structure through DNA binding, and promoting or repressing the formation of transcription factor complexes on the promoter regions of target genes (172, 173). HMGA1a expression is increased in both breast cancer cell lines and patient tumour samples. Elevated expression positively correlates with histological grade and HER2, and negatively with BRCA2 expression (174–177). Furthermore, HMGA1a overexpression in normal mammary epithelial cells promotes oncogenic transformation (174, 178).

Like CARM1, PRMT6 associates with PELP1, modulating PRMT6 enrichment of H3R2 methylation as well as PRMT6-mediated alternative splicing (160). Previously, PRMT6 was shown to mediate the alternative splicing of two genes whose expression is altered in breast cancer: vascular endothelial growth factor (VEGF) and spleen tyrosine kinase (Syk) (169). PRMT6 promotes the alternative splicing of VEGF isoform 165, the VEGF splice variant with the highest expression in breast tumour samples (179), while PRMT6 depletion inhibits splicing of a shorter form of Syk which displays increased expression in breast tumour samples compared to normal mammary epithelial tissue (180). Therefore, it is likely that PRMT6 regulation of alternative splicing, potentially through PELP1 modulation can stimulate that splicing of variants that may promote or inhibit breast cancer development.

PRMT6 methylates DNA polymerase β (Polβ), an enzyme involved in DNA base excision repair. PRMT6 methylation of Pol β arginine residues 83 and 152 is necessary to stimulate Pol β’s DNA binding capacity and facilitate its processivity (16). Low Polβ mRNA and protein expression correlates with breast cancer incidence (181, 182). This decrease in Polβ expression is associated with high tumour grade, positive lymph node status, increased ER+ tumour aggressiveness and poor patient survival (182). This suggests that PRMT6 methylation of Polβ serves as a mechanism to promote genomic stability, thus potentially inhibiting breast cancer development.

Dysregulation of the cell cycle through altered expression of critical regulatory mechanisms is often associated with oncogenesis. Inhibition of the cyclin-dependent kinase inhibitors (CDKI) p16, p21 and p27 has been shown to contribute to the development and progression of breast cancer (183–187). PRMT6 regulates the expression of these CDKIs transcriptionally or through methylation. PRMT6 depletion leads to cell cycle arrest resulting in increased expression of p16, p21 and p27 (153, 157, 188, 189), with PRMT6 methylation of H3R2 leading to transcriptional regulation of both p21 and p27 (157, 189). Furthermore, PRMT6 knockdown causes senescence in MEFs and breast cancer cells through increased p21 expression, albeit whether this occurs in a p53-dependent or independent manner remains to be determined (153, 157). Contrary to these results, Wang et al. (190) demonstrate that PRMT6 methylation of p16 impedes the propensity of the p16-CDK4 interaction that promotes cell proliferation. As discrepancies have arisen as to the precise role PRMT6 facilitates in cell cycle progression, further experimentation will be required to delineate and determine its function in the cell cycle.

Published data suggests that PRMT6 plays a significant role in breast cancer development; however, whether PRMT6 functions as a promoter or inhibitor of breast cancer remains to be determined. Discrepancies in PRMT6 expression may arise due to altered PRMT6 regulation in different breast cancer subtypes. Therefore, PRMT6 could potentially serve as a therapeutic marker in a subset of breast cancers, although further examination is required to determine which breast cancers display increased PRMT6 expression and how increased PRMT6 expression affects specific cellular pathways.

PRMT7

PRMT7 differs from the other PRMTs as it contains both N- and C-terminal putative AdoMet-binding domains. The presence of both AdoMet-binding domains is required for PRMT7 enzymatic activity; however, only the N-terminal domain binds AdoMet (191, 192). PRMT7 was initially characterised as a Type II PRMT (193), although recent data suggests that PRMT7 catalyses the formation of stable MMA; being the only PRMT proficient in catalyzing this reaction (7, 11). Though little functional data about PRMT7 is available, PRMT7 is capable of auto-methylation, and methylates substrates in a RXR motif with histone H2B an identified substrate (11). Previous characterisation of H2AR3 and H4R3 as substrates for PRMT7 (194) was likely due to contamination of the PRMT7 preparation with PRMT5 as PRMT7 is unable to methylate these arginines residues (11).

In several genome-wide studies in human patients, a strong correlation between over-expression of PRMT7 and breast cancer aggressiveness and metastasis exists. Specifically, expression of chromosome 16q22 (which contains the PRMT7 gene) is constantly upregulated in metastatic breast cancer (195). Moreover, PRMT7 interacts with CTCFL, a known oncogene which increases the activity of PRMT7 that is highly expressed in breast cancer tumours (196, 197). These studies suggest that PRMT7 may contribute to breast cancer pathogenesis, though specific experimentation still needs to be performed to identify a specific contribution of PRMT7 to breast cancer.

Small molecular inhibitors of PRMTs

Numerous groups have attempted to develop PRMT inhibitors for potential therapeutic treatment in cancer (reviewed in references 198, 199). In 2004, nine compounds named arginine methyltransferase inhibitors (AMIs) were the first specific inhibitors of PRMTs discovered. AMI-1 was shown to inhibit arginine but not lysine methyltransferase activity in vitro, prevent in vivo methylation of cellular proteins, and modulate oestrogen and androgen-mediated transcription (200). Several groups have used AMIs as analogs to synthesise additional inhibitors; however, issues pertaining to selectivity, potency, and cellular permeability have made these inhibitors less than ideal for in vivo analysis (201–204).

Pertaining to cancer, Spannhoff et al. (205) identified two compounds, allantodapsone and stilbamidine which specifically inhibit PRMT1. Both compounds were shown to inhibit PRMT1 methylation of H4R3 in a dose-dependent manner while having minimal inhibitory effects on lysine methylation of H3K4. Furthermore, both compounds could constrain estradiol-mediated activation of the ER. Bis-chloroacetyl compound 2e, a derivative of allantodapsone that specifically inhibits PRMT1 is capable of impeding proliferation in MCF7 breast cancer and LNCaP prostate cancer cells. Moreover, this compound was capable of inhibiting androgen-mediated transcription (206). Additionally, Wang et al. (207) describe inhibition of LNCaP cell growth by compound 9, a PRMT1 specific inhibitor. Lastly, Yan et al. (208) demonstrate that 2,5-bis(4-amidinophenyl)furan, a competitive inhibitor for PRMT1’s AdoMet binding site is cell permeable and can inhibit intracellular PRMT1 activity, resulting in a decrease in cell proliferation in leukaemia cells. In addition to PRMT1, specific inhibitors of CARM1 have also been identified. Cheng et al. (209) show that xenoestrogens licochalcone A, kepone, benzyl 4-hydroxybenzoate, and tamoxifen can inhibit CARM1 enzymatic activity.

Recent advances in drug development have identified compounds that can inhibit the enzymatic activity of specific PRMTs, while impeding cancer cell growth and hormone-mediated transcription. Further experimentation will be needed to determine the efficacy of these compounds in the context of breast cancer. Although the majority of compounds have focused on inhibition of PRMT1, development of additional high potency PRMT1 inhibitors, as well as inhibitors targeting other PRMTs will be needed and their efficacy in breast cancer will need to be examined.

Conclusion

The importance of PRMTs in the aetiology and progression of breast cancer is now only beginning to be examined. Altered expression and enzymatic activity of the PRMTs impact crucial cellular pathways including growth stimuli, cellular proliferation, replicative potential, apoptosis, angiogenesis and tissue invasiveness and metastasis whose differential regulation causes breast cancer development and progression (Figure 2). Thus, further experimentation should identify and elucidate the specific impact each PRMT imparts mechanistically on distinct subsets of breast tumours. In contrast to the other PRMTs, PRMT2 appears to inhibit breast tumour growth, at least in ER+ breast tumours. While limited data pertaining to PRMT3, PRMT5 and PRMT7 in breast cancer suggests that they do function in breast cell oncogenesis, further experimentation is required to determine their precise role. PRMT1, CARM1 and PRMT6 appear to promote tumorigenesis in breast cancer cells; although PRMT6 may function in only a distinct subset of tumour, these three PRMTs may serve as novel therapeutic targets in the treatment of breast cancer.

Funding

This work was supported by grants from the Cancer Research Society (JC #16135) and the Canadian Breast Cancer Foundation (JC #2013-16). Baldwin RM is a Postdoctoral Fellow of the Canadian Institute of Health Research.

Conflict of interest statement: None declared.

References