PMC

KRT80 levels are associated with changes in cell stiffness. a Magnetic tweezers (yellow) were used to measure the biomechanical properties of individual cells with or without KRT80 manipulation. Changes in cell compliance (deformation) were measured in KRT80 over-expressing cells for both parental MCF7 and AI-resistant LTED cells (left and middle graphs). Changes in cell compliance (deformation) were measured after stable KRT80 depletion in LTED cells (right graph). Significance was calculated with a student t- test and reported. b Shearwave Ultrasound measurements in prospectively recruited patients. Measures were collected at three independent location for each patient (see diagram). Plots show matched tissue stiffness for cancer vs. normal (large panel) and peri-tumoral interface vs. cancer (small inset). c KRT80 cells in diagnostic material from prospective patients assessed with ultrasound were counted using IHC. d Plots show matched tissue stiffness against the percentage of KRT80-positive cells for each individual patient. Simple linear regression was applied to calculate the correlation coefficient between these two values

KRT80-changes induce transcriptional changes of cytoskeletal genes. a PCA analyses of RNA-seq profiled MCF7 breast cancer cells or MCF7 cells with ectopic expression of KRT80. b Volcano plots of over-expressed or under-expressed genes in MCF7 cells following KRT80 ectopic expression. For a complete list, see Supplementary Data 2. c Functional enrichment for upregulated genes following KRT80 ectopic expression. d Representative confocal microscopy images showing F-actin (magenta), cortactin (CTTN, green) and DAPI (blue) staining of MCF7-control and MCF7-K80 cells. Scale bars represent 25 μm. Graph shows mean fluorescence intensity of cortactin in MCF7-control and MCF7-K80 cells (n = 40, MCF7; n = 4, MCF7-K80 individual cells). e Kaplan-Meier plot of ERα-positive breast cancer patients dichotomized to average high or low expression for genes upregulated in response to KRT80 over-expression (Panel b). Multivariate statistics are shown on the right inside table. f Current model: long-term AI treatment promotes constitutive activation of SREBP1 leading to pro-survival re-activation of estrogen receptor, and global cytoskeletal re-arrangements. Cytoskeletal re-organization leads to direct biomechanical changes and promotes pro-invasive behavior

KRT80 dynamics in treated progressing breast cancer patients. a Matched clinical specimens from breast cancer patients show an increase in KRT80 positive cells following mono-treatment (Dataset 1-ICL: Imperial College London, UK) or sequential treatment with aromatase inhibitors (Dataset 2-IEO: Istituto Europeo di Oncologia, Milan, Italy). Similar results were not significant in Tamoxifen-only treated patients. b Immunocytochemistry (IHC) analyses show changes in KRT80 protein distribution. KRT80 was imaged using IHC in a series of human samples collected at Charing Cross Hospital Imperial College NHS Trust (ICL London, UK). Tissues were collected to cover a large spectrum of benign and malignant lesions including metastatic samples from Breast Cancer patients. Yellow arrows in the bottom panels highlight cells with KRT80 expanded cytoplasmic staining. c KRT80 expression in diagnostic material has prognostic significance. Analysis were performed on the METABRIC RNA-seq splitting patient in high and low KRT80 expression. Two distinct follow-ups for an additional sub-cohort is also shown (endocrine-treated patients). Floating bars show minimum-maximum and average hazard ratios. d Other transcribed type-II Keratins in breast cancer samples from METABRIC are not associated with prognostic significance. Floating bars show minimum-maximum and average hazard ratios

KRT80 directly promotes cell invasion. a Design of the 3D invasion assay. Organoids were derived from treatment naive (green; MCF7) or invasive AI resistant (orange; LTED) breast cancer cells. KRT80 expression was manipulated via ectopic overexpression or sh-mediated stable depletion. Organoids were embedded in Matrigel and monitored for 48 h. b Representative brightfield images of KRT80-manipulated organoids. Panels show results obtained in KRT80 depleted cells. c Representative brightfield images of KRT80-manipulated organoids. Panels show results obtained in KRT80 over-expressing cells (DKK-tagged KRT80). Small inset number represent normalized fold area changes of each represented experiment. Bars scale = 400 μm. d Quantification of the area fold change in organoids overexpressing KRT80 or KRT80 knock-down LTED cells in 3D invasion assay normalized to MCF7 (*p < 0.05, **p < 0.01, Student t test; n = 3 biological triplicates in which at least 4 organoids were measured). Data is presented as mean ± SD. e Confocal microscopy of matrigel embedded invasive AI resistant LTED organoids. f Replication dependent labeling of breast cancer spheroids. Cells were labeled with CMFDA that is converted to its membrane-impermeant fluorescent form by cytosolic esterase to entrap the dye. Active replication can dilute the dye until disappearance within 2–3 cell cycles. g Quantification of the area fold change in organoids treated with CMFDA. Lines represent mean and SD. Asterisks represent significance level p < 0.05 after Student t test. h Representative images of CMFDA tagged spheroids. Invasive borders are highlighted by dotted white lines. Representative original borders are highlighted by yellow dotted lines. Bars scale = 400 μm

KRT80 induces invasion-associated cytoskeletal changes. a Representative confocal microscopy images showing F-actin (magenta), KRT80 (green) and DAPI (blue) staining of MCF7-control, MCF7-K80, LTED-control and LTED-sh^a cells. Scale bars represent 25 μm. b Zoom-up magnifications of areas indicated in a, showing F-actin (magenta), KRT80 (green) and DAPI (blue) staining in cells located at the border of clusters. Single channel images for F-actin and KRT80 are also shown. Scale bars, 10 μm. Asterisks indicate lamellipodia-like structures in MCF7-K80 and LTED cells, and hashtags indicate cortical actin areas in MCF7 and LTED-sh^a cells. Graphs on the right show line scan analysis for F-actin and KRT80 fluorescence across the leading edges of cells, as indicated in the broken line in the merged images. c, d Graphs show quantification of F-actin fluorescence intensity at lamellipodial regions (c) and at cell cortex, cytosol and overall (i.e., whole cell) (d) in MCF7-control, MCF7-K80, LTED-control and LTED-sh^a cells (n = 19, MCF7; n = 20, MCF7-K80; n = 14, LTED; n = 16, LTED-sh^a individual cells). e Graph shows quantification of percentage of cells with clear lamellipodia and membrane ruffles in MCF7-control, MCF7-K80, LTED-control, LTED-sh^a and LTED-sh^b cells (n = 8, MCF7; n = 12, MCF7-K80; n = 12, LTED; n = 7, LTED-sh^a; n = 6, LTED-sh^b fields of view). f Representative confocal microscopy images showing F-actin (magenta), pY118-Paxillin (green) and DAPI (blue) staining of MCF7-control, MCF7-K80, LTED-control, LTED-sh^a and LTED-sh^b cell. Scale bars, 25 μm. Graphs show quantification of individual leading-edge focal adhesion size (left) and number of adhesions per cell (right). Focal adhesion size (n = 269, MCF7; n = 251, MCF7-K80; n = 257, LTED; n = 331, LTED-sh^a; n = 276, LTED-sh^b). Focal adhesion number (n = 20, MCF7; n = 20, MCF7-K80; n = 20, LTED; n = 20, LTED-sh^a; n = 20, LTED-sh^b, individual cells). Statistical analyses were performed using one-way ANOVA with Tukey’s post-test. Floating bars and lines represent mean, inter-quantile distribution and SD. Asterisks represent significance at *p < 0.05, **p < 0.01 and ***p < 0.001 levels

De novo SREBP1 binding at KRT80 enhancer drives KRT80. a Predicted KRT80 enhancer clonality (x-axis) and KRT80 RNA levels (y-axis) are plotted for three independent transcriptional datasets (fantom5; HPA, Human Protein Atlas, GTEx, Genotype-Tissue Expression). Epigenetic data were obtained from the ENCODE consortia. Increasing KRT80 enhancer clonality is associated with an increasing number of KRT80 positive cells (symbols at the top, see methods for more details). b Enhancer clonality from H3K27ac data obtained in primary and metastatic breast cancer biopsies. Green dotted line indicates the presence of clonal KRT80-positive lesions. Orange dotted lines predict for KRT80-low lesions. Each circle represents an individual patient. KRT80 clonality was calculated using the core 1.5Kb H3K27ac peak. c Open chromatin profiling via DHS-seq in MCF7 and LTED cells near the KRT80 locus. d Digital Foot-printing analysis shows differential occupancy status within the E1 KRT80 enhancer. Footprint were identified using Wellington with a p < 10⁻²⁰ threshold. e ChIP-seq analysis for SREBP1 at the E1 core enhancers in invasive AI resistant breast cancer cells and treatment naive parental cell lines. SREBP1 canonical target HMGCR locus is also shown. The SREBP1 locus is bound in both MCF7 and LTED and represent the only genomic location with SREBP1 binding in parental MCF7 cells. f Stable shSREBP1 silencing in LTED cells using two independent shRNA. Individual biological replicates are shown. Lines represent means and SD. Asterisk represent significant difference at p < 0.05 after One Way ANOVA with Dunnet’s test. g Stable shSREBP1 LTED cells were assessed for SREBP1 and KRT80 protein levels

AI treatment induces KRT80 expression via epigenetic reprogramming. a Hi-C 3D interactions in GM12878 cells were analyzed using http://promoter.bx.psu.edu/hi-c/view.php. Data to derive individual TAD were downloaded from http://chromosome.sdsc.edu/mouse/hi-c/download.html. Bars represent the normalized median change in H3K27ac within the Type II-Keratin TAD compared to the overall change in H3K27ac between parental MCF7 cells (green) and drug-resistant non-invasive (gray) and drug-resistant invasive (orange) counterparts. The bottom heatmap shows the normalized expression of RNA-seq data for protein coding genes within the Type II-Keratin in all breast cancer cell lines. b Bird-eye view of the H3K27ac profile of the Type II-Keratins locus. ChIP-seq signal profiles from are shown across the entire TAD. c Targeted ChIP-qPCR for the E1 enhancer locus using H3K4me1, H3K4me2 and H3K27ac antibodies. Individual biological replicates, mean and SD are shown. Asterisks represent significance at the p < 0.001 level. d Live-imaging cell counts of mate-labeled MCF7 cells grown in presence or absence of estrogen for 48 h. Dotted line represents an ideal stalling dynamic in cell number during the time of the assay. Mean and SD of three independent counts are shown. e Population level single-cell RNA-seq data for KRT80 expression are shown. KRT80 was identified in 10.8% of MCF7 cultured in estrogen rich media and in  39.9% of MCF7 deprived of estrogen for 48 h. The distribution of the two set of data was compared using a Fisher exact test. Experiments were run comparing cells within 48 h in absence of major cell division/apoptosis. f Representative single-molecule, single cell RNA-FISH for SREBP1 (red) and KRT80 (green) in MCF7 and LTED cells. g KRT80 protein levels in MCF7 and additional independent models of invasive drug-resistant breast cancer cell lines. The asterisk represents an unspecific band. Acronyms: LTED cells: MCF7 that were long term estrogen deprived; and double resistant; MCF7T, MCF7F, LTEDT, and LTEDF: MCF7 and LTED cells resistant to Tamoxifen or to Fulvestrant respectively; ETR: endocrine treatment resistant; E2: estrogen; TAD: topological associated domain; RPKM: reads per kilobase million; KRT: keratin; SREBP1: sterol regulatory element binding protein 1