SREBP1 drives KRT80-dependent cytoskeletal changes and invasive behavior in endocrine resistant ERα breast cancer

Approximately 30% of women diagnosed with ERα breast cancer relapse with metastatic disease following adjuvant treatment with endocrine therapies1,2. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain (TAD)3 undergoes epigenetic reprogramming in cells that develop resistance to aromatase inhibitors (AI), leading to keratin 80 (KRT80) upregulation. In agreement, an increased number of KRT80-positive cells are observed at relapse in vivo while KRT80 expression associates with poor outcome using several clinical endpoints. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 14 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion maturation and cellular stiffening, which collectively promote cancer cell invasion. Shear-wave elasticity imaging of prospective patients shows that KRT80 levels correlate with stiffer tumors in vivo. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

H3K27ac and H3K4me1-2 5,9,10 . These epigenetic changes occasionally involve entire topological associating domains (TADs), three-dimensional compartments within the genome thought to restrict enhancer-promoter interactions 3,11 . We observed that the type II keratin TAD ranked among the most significantly epigenetically reprogrammed (top 5%) when comparing untreated, non-invasive parental vs. invasive AI resistant BC cell lines 5 (long term estrogen deprived: LTED cells, Figure 1A). Type I and Type  Figure 1A and S1A-B). Measuring KRT80 transcripts before or after shortterm (48hrs) acute estrogen starvation using single cell RNA-seq data we showed that the significant increase in KRT80 positive cells is driven by transcriptional activation and not selection of KRT80-positive clones ( Figure 1B). These data were validated in MCF7 and LTED cells using single cell RNA-FISH ( Figure 1C). As expected, increased transcription corresponded to increased KRT80 protein level ( Figure 1D).
KRT80 is a largely unknown keratin structurally related to hair keratins 12 , in contrast with epithelial keratins commonly found in normal epithelial cells. This led us to further explore the role of KRT80 in promoting the aberrant phenotype observed in AIresistant cells. KRT80 transcripts were also elevated in several ERα-negative cell lines, suggesting that upregulation in drug-resistant cells was not mediated by changes in ERα activity (Table S1). More importantly, IHC analysis of two independent clinical datasets confirmed that KRT80 positive cells increase after AI treatment but not Tamoxifen in vivo 13,14 (Figure 1E). KRT80 localization in vivo was radically different to what has been shown in conventional keratins (e.g. KRT8, KRT14, KRT18 or KRT19 15 ), presenting a peri-nuclear polarized pattern towards the lumen within healthy ducts and lobules (Figure 1F and S2). Similar staining patterns were conserved in benign lesions (Figure S2), whereas KRT80 staining became strongly cytoplasmic in higher grade BC and metastatic lesions suggesting a potential role in BC progression ( Figure 1E). Correspondingly, high KRT80 mRNA levels correlated with poor survival in the TCGA-BC dataset (Fig. 1G). These data were confirmed by multivariate metaanalysis of two independent datasets with additional clinical endpoints ( Figure S3).
Activation of cell type specific enhancers has been linked with cancer transcriptional aberration 9,16-18 , leading us to hypothesize that de novo enhancer activation within the TAD structure might control KRT80 expression in AI resistant cells. We used H3K27ac, an epigenetic mark associated with gene activation 9,19 , to identify KRT80 potential enhancers (E1 and E2, Figure S4A). As expected, E1-E2 activity was only captured in KRT80-positive cells ( Figure S4A). 3D meta-analysis from parental MCF7 ChIA-Pet data strongly suggested that the E1 loci could contact the KRT80 promoter via enhancer-promoter interactions, while it excluded the weaker E2 ( Figure S4B) suggesting that the 3D interaction is already pre-establihed in sensitive cells. To test whether E1 drove KRT80 transcriptional activity we adapted our recently developed computational pipeline to measure the relative size of KRT80positive clones in several tissues 9 . Analysis of Epigenetic Roadmap data 20 strongly suggested that E1 activity controls KRT80 transcription levels ( Figure 1H). E1 activity was also potentially associated with KRT80 transcription in several cell lines ( Figure   S4C-D). KRT80 E1 activity also correctly predicted strong expression in mammary epithelium cells (Figure S4C-D). Finally, E1 enhancer activity analysis predicted a significant increase in KRT80 positive cells in AI resistant models, in agreement with mRNA and protein analysis ( Figure S5). Using fine-mapping analysis we identified a core-region within the E1 enhancer (1.5Kb) more strongly associated with KRT80 expression in our BC cell lines ( Figure. S5). This core enhancer showed a clear pattern of activity in actual BC patients 9 predicting the existence of KRT80 clonal and sub-clonal population in primary and metastatic BC ( Figure. 1I). Overall, these data suggest that core-E1 is the critical enhancer driving KRT80 expression in BC cells.
We next investigated which transcription factor/s (TFs) might regulate KRT80 expression. DHS-seq analysis 5 indicated that KRT80 is already accessible in MCF7 ( Figure. 2A), yet digital foot-printing suggested different occupancy rates ( Figure. 2B). More specifically, we noted the appearance of a SREBP1 footprint within the core-E1 unique to LTED cells. We have previously reported that AI resistant cells upregulate lipid biosynthesis via global epigenetic reprogramming 5 suggesting widespread SREBP1 activation in AI resistant cells. ENCODE TFs mapping showed that SREBP1 bind the core-E1 enhancers in lung cancer cells, the only ENCODE profiled cells characterize by strong KRT80 transcription ( Figure. S6A-B). To directly test if SREBP1 drives KRT80 expression in BC we performed ChIP-seq in MCF7 and T47D cells and their respective AI-resistant models. Our data demonstrate that SREBP1 was bound at core-E1 only in AI-resistant BC cells ( Figure. 2C and S6C).
Interestingly, the expression of KRT80 and SREBP1 target genes was also strongly correlated in BC patients ( Figure. S6D). Finally, we show that SREBP1 silencing abrogated KRT80 expression in LTED cells ( Figure. 2D). Overall these data demonstrate an unpredicted link between SREBP1 and KRT80 activation. Intriguingly, the footprint containing the SREBP1 motif was not under significant evolutionary constraint ( Figure.  Several studies have investigated how mechanical stimuli influence the epigenetic landscape 21,22 . However, our data implied a novel causal link whereby epigenetic reprogramming promoted changes in specific cytoskeletal components (e.g. KRT80) which may ultimately affect the biophysical properties of cells and tumors 23,24 (Figure 1-2). In agreement, we observed a significant increase in cellular stiffness (inversely correlated to cell compliance) after KRT80 over-expression in MCF7 and LTED cells ( Figure 2E). To test if KRT80 can contribute to tumor stiffness in vivo we prospectively recruited 20 patients with suspected BC and performed shearwave elastography to measure intra-tumoral stiffness ( Figure 2F). Our data showed that cancer lesions had significantly higher stiffness than surrounding normal tissues, with the highest peak of stiffness consistently measured at the invasive border ( Figure   2F). Interestingly, meta-analysis of tumor and matched nearby tissue from TCGA show increased KRT80 mRNA in the tumor biopsies ( Figure 2F). We then performed IHC for KRT80 with validated antibodies (Figure 2G and S8) using biopsies collected from our prospective patients. Linear regression analysis showed that KRT80 positivity significantly correlated with intra-tumor stiffness (Figure 2G and S8). Collectively, these data demonstrate that BCs characterized with high KRT80 content are mechanically stiffer.
The effect of increasing stiffness in metastatic invasion is highly debated.
Previous studies have suggested that decreased stiffness, through loss of keratins, improves single-cell invasion 23 typical of EMT cells. However, solid tumors can also use a myriad of multicellular invasion programs 25 collectively termed "collective invasion". Recent studies have shown that keratins such as KRT14 can play critical roles in collective invasion 26 6 and multi-clonal metastatic seeding 26 , two processes driving BC progression 26 . In addition, a significant body of clinical literature has linked increased breast tumor stiffness to poorer prognosis 26 and lymph node positivity 26 , independently of changes in extracellular matrix stiffness. We reasoned that a model in which KRT80 upregulation in BC cells leads to increased stiffness and augmented collective invasion might reconcile all these observations. To test this, we developed 3D spheroids from MCF7 or LTED cells and assessed collective invasion ( Figure 3A) after KRT80 manipulation (Figure S9A-D). Spheroids from KRT80-positive LTED cells could effectively invade intro matrigel matrices, but KRT80 depletion completely  To test if KRT80 manipulation drives ancillary phenotypes synergistic to cytoskeletal changes, we performed RNA-seq in cells transfected with KRT80 but where SREBP1 is not yet activated (non-invasive MCF7 cells, Figure 4A). Ectopic   Together, these results further support that KRT80 manipulation is sufficient to activate genes driving dramatic cytoskeletal rearrangements that ultimately induce invasive behaviors in BC and poorer prognosis. We cannot speculate at the moment if this is driven by a cytoskeleton-transcriptional feedback or is mediated by some specific

Tissue specimens
Seventy-five human breast specimens and ten metastatic lymph nodes were selected from Histopathology Department at Charing Cross Hospital, with the previous approval of Imperial College Healthcare NHS Trust Tissue Bank.
A Tissue Microarray (TMA) containing 26 primary breast tumors and paired ETR relapses was constructed as previously described (18).
Immunohistochemistry staining was scored using a quick score system by two independent investigators, one of them a consultant pathologist (SS). Score was calculated as follows: S=3 (strongly stained cells), S=2 (moderate staining), S=1 (poorly stained cells) and S=0 (absence of staining). Staining intensity was assessed as mean intensity from the tumor region contained within the TMA.

Immunohistochemistry
Formalin fixed and paraffin embedded (FFPE) tissue specimens were sliced in 4 µm minutes in running tap water and following that, nuclei was stained with haematoxylin.
Slides were dehydratated in 100% ethanol, cleared in xylene and mounted in DPX (SIGMA).

Statistical analysis
Data is presented as mean ± SD (standard deviation). Data analysis was performed using GraphPad Prism 6 software. An unpaired two-tailed Student's t test was applied to all data, except from a non-parametric Mann-Whitney test applied to TMA score.

Survival analysis
Publicly available breast cancer datasets were identified in GEO

Shearwave Elastography
All SWE was performed by a breast radiologist with more than 10-years experience of performing Breast ultrasound and elastography on breast lesions. A state-of-the art ultrasound scanner, Aplio i900 (Canon Medical Systems, Nasu, Japan) with the latest 2D SWE technology was used for this study. All SWE maps and calculations were obtained pre-biopsy. A good stand-off was used for superficial lesions and initially, continuous SWE mode ("multi-shot") was used to select the optimum plane and once this was stabilised, a higher energy SWE push-pulse ("one-shot" mode) was then utilised to obtain the final elastogram for calculations. Regions of interest (ROI) were placed within the centre of the lesion, in the periphery and also within the adjacent normal breast tissue. This has been stored as raw data within the ultrasound systems which would enable any re-calculations as necessary.

Supplementary Figures
Supplementary Figure 1 Manipulation of KRT80 directly reprogram these sub-structures.