Surgery (St Vincent's)

Pre-mRNA alternative splicing in the epithelial to mesenchymal transition of breast cancer cells Edwin Widodo, Eva Tomaskovic-Crook, Bryce van Denderen, Erik W. Thompson Summary Alternative pre-messenger RNA splicing is a process that generates multiple variants of a single gene by virtue of the alternative exons that are transcribed. In breast cancer progression and metastasis, alternative splice events (ASE) are regulated during epithelial to mesenchymal transition (EMT). EMT occurs naturally during embryonic development as epithelial-derived cells become transiently mesenchymal and move around the embryo to generate the body plan. EMT status can be determined by expression of specific markers for EMT. E-cadherin is recognized as the archetypical marker for the epithelial phenotype. During carcinoma EMT, E-Cadherin is reduced by transcriptional repression and/or translocation away from the membrane junctions, and the cytokeratin intermediate filament network is reduced or lost while vimentin expression is enhanced. EMT manipulation can be implemented by inducing overexpression of EMT-regulating driver genes, including Twist1 and Snail1 (Mani et al., 2008), and is prominently driven by transforming growth factor beta (TGFbeta). These genes act by transcriptional repression of E-Cadherin. The nuclear factor kappa B (NF-kB) pathway has also been shown to be involved in EMT in MCF10A breast cancer cells. Human breast cancer cell lines are mostly divided into 5 categories based on their characteristics defined in clinical breast cancer datasets. The categories are Luminal A, Luminal B, Basal A, Basal B, and HER2+ types. Hierarchical clustering of high throughput array studies conducted on 34 (Charafe-Jauffret et al., 2006) and 51 (Neve et al., 2006) measuring RNA expression on human breast cancer cell lines grouped those cell lines into Luminal and Basal subgroups. Luminal cells often express estrogen receptor (ER+) and progesterone receptor (PR+) while Basal cell lines lack expression of ER, PR and HER2 (triple negative) and are more resistant to adjuvant chemotherapy. The Luminal group was further divided into Luminal A with low Ki67, a marker of proliferating cells, and Luminal B with high Ki67. The Basal group of cell lines was further divided into 2 groups, Basal A and Basal B (Neve et al., 2006). Most cell lines in the Basal B group have a more invasive phenotype and exhibit a mesenchymal gene signature. We applied a panel of cell lines from those different molecular subgroups: Luminal (MCF7, which has epithelial features), Basal A (MDA-MB-468) and Basal B (MDA-MB-231, which has mesenchymal properties). The EMT features in the PMC42 system include down-regulation of CDH1 and up-regulation of Vimentin for PMC42-ET and PMC42-LA cell lines (Ackland et al., 2001, Ackland et al., 2003). PMC42 system consists of the parental PMC42-ET and the epithelial subtype, PMC42-LA, the PMC42 system provides us with a spectrum of EMT associated changes. The PMC42-LA cell line contains a low number (10-15%) of Vimentin-positive cells, whereas the PMC42-ET cells are 100% Vimentin positive, with commensurate CDH1 differences (Hugo et al., 2007). Further, in response to EGF PMC42-LA cells undergo EMT-like changes (Ackland et al., 2003). ASE in the PMC42 human breast cancer EMT were investigated by (i) comparisons between the more mesenchymal parental PMC42-ET (ET) cells and the more epithelial PMC42-LA (LA) subline, and (ii) in response to epidermal growth factor (EGF), which stimulates EMT-like changes at the mRNA and protein level in both PMC42 variants. We assessed these effects in 2D monolayer culture as well as 3D cultures in Matrigel or Collagen (Vitrogen) and found very similar results in all three culture conditions. ASE are regulated by Epithelial Splice Regulatory Proteins (ESRP) 1 and 2. The expression of both, ESRP1 and ESRP2, was found to be suppressed in Basal B but not in Luminal or Basal A cell lines (Warzecha et al., 2009a). This suggests that their suppression could be involved in EMT-related events. ESRP1 and 2 mRNA levels were constitutively lower in the mesenchymal ET cells compared to LA, but showed little EGF regulation. ESRP1 mRNA levels in epithelial MCF-7 cells were similar to LA, while mesenchymal MDA-MB-231 cells were similar to ET. For ESRP2, MCF-7 levels were higher than LA. Mammalian Ena Homolog (MENA) levels in both PMC42 variants resembled MCF-7 cells, however both variants predominantly expressed the mesenchymal-associated form, as was the case with Cluster of Differentiation 44 (CD44), Ral GEF with PH Domain and SH3 Binding Motif 2 (RALGPS2) and Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1 (MAGI1). Thus, EMT-associated-ASE revealed predominantly mesenchymal-specific splicing patterns despite the ESRP1 differential, perhaps due to a lack of ESRP2. The results confirm an ESRP1/2-related mesenchymal shift from PMC42-LA to ET cells. In general, we also confirmed a high level of ESRP1 and Fibroblast growth factor receptor 2 – exon IIIb (FGFR2 IIIb) in Luminal and Basal A cells, and reduced level of ESRP1 and higher Fibroblast growth factor receptor 2 – exon IIIc (FGFR2 IIIc) in Basal B cells. The shift from FGFR2-IIIb to FGFR2-IIIc in EMT showed alternatively spliced variants from the same gene, FGFR2. Expression of FGFR2-IIIb measured by splice-specific RT-PCR followed the same pattern as ESRP1, while both PMC42 variants were higher than MCF-7 cells for mesenchymal-associated FGFR2-IIIc. Zinc finger E-box-binding homeobox 1 (ZEB1) mRNA levels, a transcription factor that binds E-box motifs in promoters, were reduced by expression of a short hairpin RNA (shZEB1) in PMC42-ET cells. Lack of ZEB1 resulted in a significant reduction (p=0.0018) of ESRP1, but not ESRP2, consistent with the E-box in the ESRP1 proximal promoter. Although FGFR2 IIIb was upregulated after ZEB1 silencing (p=0.058), FGFR2 IIIc, which was supposed to be alternatively spliced, remained at the same level after ZEB1 silencing (p=0.6263). This suggests a direct role of ZEB1 in ESRP1 expression. Total Enabled Homolog (ENAH) was not reduced significantly after ZEB1 knockdown, (p=0.366). In summary, ZEB1-knockdown in PMC42-ET cells caused enhanced levels of ESRP1 and FGFR2 IIIb expression. Partek Genomic Suite analysis of the Affymetrix data indicated a selective upregulation of a 3’-truncated isoform of Laminin subunit alpha 3 (LAMA3 variant 2 or LAMA3v2) by EGF. qRT-PCR analysis revealed that both the long variant (LAMA3v1) and shorter variant (LAMA3v2) showed enhanced levels along the Luminal to Basal B spectrum (as explained in Chapter 1.1.3.), although the Basal B MDA-MB-231 cells appeared to under-express LAMA3v2. LAMA3v2 was particularly highly expressed in the PMC42 variants and was upregulated by EGF in PMC42-LA cells but not in the PMC42-ET cells. LAMA3v1 levels in PMC42-LA and –ET cells both resembled the MDA-MB-231. In the MDA-MB-468 model of EGF-induced EMT, LAMA3v1 was stimulated by EGF treatment (7 days) but not hypoxia (3 days), whereas LAMA3v2 expression was stimulated by either EGF (7 days) or hypoxia (3 days) treatments. Our group has conducted an experiment by xenografting MDA-MB-468 in mice. In MDA-MB-468 xenografted tumours, LAMA3v2 was expressed significantly higher than LAMA3v1. Gene silencing using small interference RNA (siRNA) techniques provide a sight on the short term effects of knocking down gene(s) in cells. In assessing the effectiveness of silencing subunit alpha 3, subunit beta 3 and subunit gamma 2 of Laminin (LAMA3, LAMB3 and LAMC2), we used short interference RNA (siRNA) targeting LAMA3 (siLAMA3), LAMB3 (siLAMB3) and LAMC2 (siLAMC2) and combination of those three siRNAs (siLAMA3B3C2). On the one hand, inhibition of LAM using the siLAMs, as confirmed with inhibition of LAMB3 LAMC2, and laminin v2, inhibited the expression of EMT markers: Vimentin. On the other hand, siLAM increased expression of Zeb1, ENAH and laminin v1. This may suggest that targeting Laminins using siRNA could reduce the EMT properties of PMC42 cell lines. RNA Seq results showed several top genes as potential candidates for EMT in PMC42 breast cancer cells. We applied Multivariate Analysis of Transcript Splicing (MATS) and Differential Exon Usage in RNASeq (DEXSeq) to identify several top candidates, which have upregulated transcript variants during EMT in the PMC42 system. Those top candidates were inspected using SeqMonk to visualize RNASeq reads. Several transcripts were listed on MATS and DEXSeq results as having one of their exon upregulated after EGF treatment in PMC42-LA, including Ladinin-1 (LAD1), Tenascin-C (TNC), Cleft Lip and Palate Transmembrane Protein-1 Like (CLPTM1L), Serine / Arginin-rich Splicing Factor 1 (SRSF1). In this investigation, only LAD1 showed a pronounced up-regulated transcript variant in SeqMonk visualization. Thus, LAD1 is a good candidate as a target for inhibiting EMT in PMC42 breast cancer cell line.

Breast cancer is the most common malignancy among women worldwide, with mortality primarily associated with metastasis. In recent years, microRNAs (miRNAs) have emerged as a new class of master regulatory molecules with the potential to influence carcinoma progression. They are a class of small RNA molecules that regulate gene transcript stability and processing by binding to discreet motifs in the 3’ and 5’ untranslated regions of mRNAs. They regulate important mechanisms in development, including epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have also been associated with cancer metastasis. miRNA profiling of control and EMT-induced PMC42-ET, PMC42-LA and MDA-MB-468 cultured human cell lines using microarray and Next Generation Sequencing (NGS) was undertaken. Several miRNAs were reproducibly up- or downregulated between the untreated cells, and in response to epidermal growth factor. Variations in miRNA expression were also assessed bioinformatically using publically available data from >50 human breast cancer cell lines. Whilst a number of these have already been implicated in cancer, other novel miRNAs consistently associated with EMT/MET were also identified. The expression levels of >20 miRNAs were experimentally validated and stable miRNA manipulations (overexpression and knockdown) in cell lines with low endogenous expression were achieved by lentiviral transduction. Recent evidence has demonstrated a crucial role for the miR-200 family in carcinoma progression, tumourigenesis and metastasis in various cancers including breast cancer. miR-200 regulation of EMT and MET was identified in our microarray and NGS datasets and was characterised in the two EMT models commonly used in our laboratory – the PMC42 and MDA-MB-468 human breast cancer cell lines. In vitro functional changes upon miR-200 manipulation showed a strong correlation between high miR-200 levels and the epithelial phenotype. In vivo changes mirrored the in vitro changes, where miR-200c knockdown resulted in reduced primary tumour growth and increased axillary lymph node metastasis in the MDA-MB-468 xenograft model. Transcriptionally, it was observed that the MDA-MB-468 miR-200c knockdown tumours showed upregulation of epithelial genes (such as E-cadherin, Grhl2, EpCAM) and a corresponding downregulation of mesenchymal genes (such as BNIP3, FN1), suggesting that the MDA-MB-468 miR-200c knockdown cells that were able to survive and eventually form a tumour had to transcriptionally activate epithelial-associated genes to achieve tumour development. Despite this, circulating tumour cells in the peripheral blood of MDA-MB-468 miR-200c knockdown tumour-bearing mice showed indications of a hybrid state, trending towards a more mesenchymal profile. Apart from the miR-200 family, eight other miRNAs—namely miR-744, miR-153, miR-708, miR-483, miR-100, miR-34b/c, miR-146a and miR-29a, were short-listed as candidate EMT-associated miRNAs identified from microarray, NGS and bioinformatic analyses. Collectively our findings suggest that no one particular miRNA alone was capable of functionally altering MDA-MB-468 breast cancer cells. Interestingly, when the cells were driven to a more mesenchymal state, there was a trend towards increased lymph node metastasis, suggesting that a mesenchymal profile is advantageous in that process. The overall findings of this thesis suggest that miRNAs do play a key role in the area of breast cancer EMT and each step in the metastasis cascade can be regulated by different miRNAs. A cooperative network of miRNA changes results in downstream changes in tumourigenesis and metastasis. These studies highlight the need to further investigate context-dependent miRNA manipulations and validate miRNAs that play critical regulatory roles to properly understand the complex role of various important miRNAs in breast cancer EMT.

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