Abstract
Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)–Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK–Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell’s environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.
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Data availability
The data supporting the findings of this study are available from the corresponding authors on reasonable request. All relevant data used to generate Figs. 1–5 and Extended Data Figs. 1–7 are included in the paper as Source Data. RNA-seq data from this study have been deposited in the GEO database under accession number GSE128037.
Change history
25 February 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41563-020-0644-5
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Acknowledgements
We thank V. Guzzardo for histology; C. Frasson for FACS; M. Ventre and P. Netti for rheology; E. Armato Smaniotto dai Roveri for human bioptic sample procurement; and D. J. Pan, F. Camargo, G. Lozano, T. Jacks and J. Siveke for gifts of mice. A.C. was supported by a CARIPARO PhD fellowship. The research leading to these results has received funding from the following agencies: the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (DENOVOSTEM grant agreement No. 670126) to S.P.; Fondazione AIRC under the 5 per Mille 2019 programme (ID No. 22759) to S.P.; the Italian Ministry of Education, University and Research (MIUR), MIUR-FARE (No. R16SXW55W4) to S.P. and PRIN 2017 grants to T.P. (No. 2017L8FWY8_004) and S.P. (No. 2017HWTP2K_001); Fondazione CARIPARO, under the CARIPARO Starting Grant programme (No. C94I19001680001) to T.P. and a CARIPARO Ricerca Scientifica di Eccellenza 2018 grant (No. 52008 - 2019.0356) to S.P.; and University of Padua PRID grant to S.P. and L.A. (No. CPDA135844).
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Contributions
T.P. and A.C. performed all in vitro and in vivo experiments. A.R. helped with mammary gland transplantation. T.P and D.D.B. performed human mammary gland experiments; S.G., G.Br. and A.G. performed hydrogel preparation; L.A. performed mouse genetics; M.F., M.C. and S.B. performed bioinformatic analysis; V.P. and S.F. performed microrheology and TFM; M.F. performed histology and histopathological evaluations; and V.V., F.B., M.F. and A.P.D.T. managed human samples. T.P., M.C. and S.P. conceived the initial hypothesis and experimental design, and planned, discussed and organized the work. T.P., A.C., M.C. and S.P. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Abnormal substrate rigidity is required for oncogenes to reprogram normal cells into tumorigenic ones.
a, Schematic representation of the FACS purification strategy and of the experiments performed with mLD cells. LP: luminal progenitor cells. b, qRT-PCR analyses for the indicated basal and luminal mammary cell markers in mBasal, mLP, and mLD cells obtained by FACS, as in Extended Data Fig. 1a. Data are normalized to Gapdh and are referred to Basal cells levels for basal genes, and to LD cells levels for all the other luminal markers (each set to 1). Results are representative of n=3 independent experiments (each using mammary glands from n=20 mice) performed with similar results. c, Schematic representation of the FACS purification strategy and of the experiments performed with human primary mammary luminal cells. d, qRT-PCR analyses for the indicated basal and luminal mammary cell markers in human Basal, LP, and LD cells obtained by FACS, as in Extended Data Fig. 1c. Data are normalized to GAPDH and are referred to Basal cells levels for basal genes, and to LD cells levels for all the other luminal markers (each set to 1). Results are representative of n=3 independent experiments performed with similar results. e–h, Lower magnification images of experiments shown in Fig. 1a (e), in Fig. 1c (f), in Fig. 1f (g) and in Fig. 1g (h). Scale bars, 600 µm. i, Representative bright field and immunofluorescence images (n=3 independent experiments) of organoids formed by mLP cells expressing HER2-CA. K14 serves as marker of basal cell identity and K8 serves as marker of luminal cell identity. Scale bars, 400 μm, left and 17 μm, right. j, Quantifications of the frequency of different outgrowths emerging from fat pad injection of the same samples of Fig. 1i. Data are representative of n>5 independent samples. k, Representative GFP-immunofluorescence pictures (n>5 independent samples) of outgrowths depicted in Fig. 1i, showing that outgrowths emerge form injected mLD cells. Scale bar, 50 μm. l, Representative immunofluorescence images (n=3 independent experiments) of ex-vivo 3D outgrowths formed by mLD cells expressing HER2-CA, embedded in a collagen-based ECM and treated with conditioned media from Empty-vector (CM: Empty) or LOXL2-transduced C3H10T1/2 fibroblasts (CM: LOXL2). While HER2-expressing cells formed round spheroids/organoids in absence of LOXL2, those embedded in LOXL2-modified collagen formed highly proliferating and invasive outgrowths. This effect relied on LOXL2 enzymatic activity as it was abolished by concomitant treatment with LOX-inhibitor BAPN. Scale bars, 260 µm. m, Bright field and birefringence Picrosirius Red images of the same samples shown in Fig. 1i, showing LOXL2 mediated increased fibrillar collagen deposition. Scale bars, 100 µm. Quantifications of total fibrillar collagen by birefringence signal in the same sections are presented as mean + s.d. of n=4 independent samples. ** p-value=7.13x10-3. n, Representative images and quantifications of colonies formed by mLD cells transduced with the indicated constructs. The expression of the sole integrinβ1 mutant is inconsequential for colony formation by mLD cells. Scale bars, 170 μm. Images and data are representative of n=4 independent experiments. Data are mean + s.d. **** p-value=4.03x10-6. o, Low magnification of the representative histological staining of the subcutaneous outgrowth shown in Fig. 1j (corresponding to the frame). P-values were calculated by one-way ANOVA with Sidak’s multiple comparisons test.
Extended Data Fig. 2 Ras/RTK oncogenes change the mechanical and material properties of cells.
a, Representative images and quantifications of collagen contraction assays performed with control MCF10A cells transduced with empty vectors and embedded in 3-D collagen hydrogels with conditioned media obtained from control MCF10A cells (CM: Empty) or cells expressing KrasG12V (CM: KrasG12V) or HER2-CA (CM: HER2-CA). Compared with Fig. 2a, this shows that oncogene-mediated cell contractility does not rely on secreted factors. Images and data are representative of n=3 independent samples. Data are mean + s.d. b, Quantification of the colocalization of the cytoskeletal stainings for pMLC and F-actin shown in Fig. 2c. Data are as mean + s.d. of n=3 independent samples. c, Representative immunofluorescence images and quantifications of FA formation by colocalization of focal adhesion kinase (FAK) and Vinculin in MCF10A cells transduced with HER2-CA or KRasG12V. Scale bars, 24 μm. Images and data are representative of n=3 independent experiments. Data are mean + s.d. d, Representative immunofluorescence images and quantifications FA maturation by colocalization of phospho-focal adhesion kinase (pFAK) and Paxillin in MCF10A cells transduced with HER2-CA or KRasG12V. Scale bars, 24 μm. Images and data are representative of n=3 independent experiments. Data are mean + s.d. e, Representative pictures (n=3 independent experiments) and quantifications of proximity ligation assay (PLA) detecting the nuclear interaction between endogenous YAP and endogenous TEAD37 in control (Empty vector) MCF10A cells or in cells overexpressing KrasG12V or HER2-CA. Scale bars, 9 µm. Data are mean + s.d. of n>200 cells. **** p-value=10-15. f, Luciferase assay in post-confluent HEK293 cells transfected with a synthetic reporter for TEAD-dependent transcription (8xGTIIC-Lux) and with the indicated siRNAs and control (Empty) or oncogene-expressing vectors. Data are mean + s.d. of n= 3 independent samples. ****p-value=10-15. g, qRT-PCRs of the YAP/TAZ endogenous targets CTGF and AXL, in MCF10A cells transduced with the indicated oncogene-expressing vectors. Data are mean + s.d. of n= 3 independent samples. h, Collagen contraction assays performed with MCF10A cells transduced with the indicated vectors, transfected with control or YAP/TAZ targeted siRNAs, showing that YAP/TAZ are downstream of cell contractility induced by oncogenes. Data are mean + s.d. of n= 3 independent samples. Immunoblots are shown for validation of effective YAP/TAZ depletion. GAPDH serves as loading control. i, Representative immunofluorescence images and quantifications of YAP/TAZ localization in MCF10A cells transduced with empty or oncogene-expressing vectors, and seeded on 40 kPa hydrogels. Scale bars, 8 μm. Data are mean +/- s.d. of n>17 independent samples. j, qRT-PCRs of the YAP/TAZ endogenous target CYR61 in MCF10A cells transduced with the indicated vectors and seeded on 0.5 or on 1 kPa hydrogels, as in Fig. 2g. Data are mean + s.d. of n= 3 independent samples. k, Representative immunofluorescence images of F-actin of the same MCF10A cells shown in Fig. 2f to visualize cell shape. F-actin was stained with fluorescently-labelled phalloidin and nuclei were counterstained with DAPI. Scale bars, 16 µm. P-values were calculated by unpaired two-sided Student’s t-test (a), one-way ANOVA with Sidak’s multiple comparisons test (b–f) or two-way ANOVA with Sidak’s multiple comparisons test (g, h, j).
Extended Data Fig. 3 Oncogenes empower a disproportional cellular response to ECM mechanical properties to drive pancreatic tumorigenesis.
a, Schematic representation of the experimental strategy employed to study pancreatic ADM ex vivo. See also methods section for details. b, Schematic representation of the chemistry of designer hydrogels employed in the experiments of Fig. 3a–c, Fig. 4c, Extended Data Fig. 3c and Extended Data Fig. 4e, f (see Methods). SH, sulfhydryl group; PEGDA, Poly(ethylene glycol) diacrylate. Hydrogel stiffness was raised by increasing the PEGDA crosslinker concentration, without changing Gelatin and Hyaluronic Acid content. c, Quantification of nuclear YAP/TAZ levels in pancreatic acini shown in Fig. 3c. Data mean + s.d. of n=4 independent samples. ****p-value=4.03x10-7. d, qRT-PCRs assessing the expression levels of the acinar marker Amylase (Amy) and of the ductal markers K19 in pancreatic acini of the indicated genotypes, showing that KRas-expressing acini treated with FA or F-actin inhibitors (as in Fig. 3d) remain fully differentiated recapitulating the effects of a soft ECM. Data are mean + s.d. of n= 3 independent experiments. ns, p-value=0.99. Data are normalized to 18-s rRNA. e, Quantification of nuclear YAP/TAZ levels in pancreatic acini shown in Fig. 3d, showing that oncogenic KRas promoted YAP/TAZ activation in a manner opposed by FA and F-actin inhibitors. Data are mean + s.d. of n>4 independent samples. **** p-value=10-15. f, Dot-plot depicting cell viability assays comparing MCF10A cells treated with the indicated mechano-inhibitory drugs (at maximal doses, see methods for details) with untreated cells. Drug treatments employed throughout the study do not affect cell viability, at difference with Puromycin (Puro.) treatment, shown as positive control for reduced cell viability. Data are mean +/- s.d. of n=5 independent samples. g, Schematic representation of the experimental strategy employed to oppose either ECM stiffness (by BAPN treatment) or intracellular contractility (by Fasudil treatment) at the onset of pancreatic tumorigenesis in vivo. h, Quantifications of tumor areas in the samples depicted in Fig. 3f. Data are mean +/- s.d. of n=3 independent samples. i, Quantifications of pMLC stainings shown in Fig. 3f, showing that BAPN treatment normalized the tensional state of oncogene-expressing pancreatic cells. Data are mean + s.d. of n=3 independent samples. ***p-value=6.6x10-4. j, Representative bright field and birefringence images of Picrosirius red staining of the same samples shown in Fig. 3f, showing that BAPN treatment inhibited deposition and fibrillar organization of collagen. Quantifications of total fibrillar collagen obtained from birefringence are presented as mean + s.d. of n=3 independent samples. ****p-value=1.80x10-5. k, Quantifications of tumor areas in the samples depicted in Fig. 3g. Data are mean +/- s.d. l, Quantifications of pMLC stainings shown in Fig. 3g. Data are mean + s.d. of n=3 independent samples. ****p-value=8.88x10-5. P-values were calculated by unpaired two-sided Student’s t-test (c) and by one-way ANOVA with Sidak’s multiple comparisons test (d–f, g, i, l).
Extended Data Fig. 4 YAP/TAZ are the nuclear effectors downstream of the changes in the cell’s mechanical and material properties induced by oncogenes.
a, Representative H&E stainings and quantifications of tumor areas showing that initiation of pancreatic tumorigenesis form acinar cells is dependent on YAP/TAZ. Development of neoplastic lesions was assessed 6 months after tamoxifen administration to mice of the following genotypes: Ptf1aCreERTM(wt), Ptf1aCreERTM;KRas+/LSL-G12D(KRasG12D), Ptf1aCreERTM;KRas+/LSLG12D;Yapfl/fl;Tazfl/fl (KRasG12D;Y/T KO), Ptf1aCreERTM;KRas+/LSLG12D;p53+/LSLR172H (KRasG12D;p53R172H), Ptf1aCreERTM;KRas+/LSLG12D;p53+/LSL-R172H;Yapfl/fl;Tazfl/fl(KRasG12D;p53R172H;Y/T KO). Data are presented as mean +/- s.d. of n=3 independent samples. These experiments are similar to what reported in Ref. 23, although in a different experimental setting, as we tested Yap/Taz requirement in absence of experimentally induced pancreatitis. b, Immunoblot validating effective Yap and Taz in vivo knock out in pancreata shown in a. GAPDH serves as loading control. Results are representative of n=3 independent experiments. c, Quantifications of the percentage of ADM events in pancreatic acini depicted in Fig. 4b. Data are presented as mean + s.d. of n=5 independent samples.****p-value=10-15. d, qRT-PCRs validating effective Yap and Taz ex vivo knock out in pancreatic acini shown in Fig. 4b. Data are presented as mean + s.d. of n=3 independent samples. Data are normalized to 18-s rRNA. e, Quantifications of the percentage of ADM events in pancreatic acini depicted in Fig. 4c. Data are presented as mean + s.d. of n=3 independent samples. f, qRT-PCRs validating YAPS127A overexpression in pancreatic acini shown in Fig. 4c. Data are mean + s.d. of n=3 independent samples. Data are normalized to 18-s rRNA. g, Quantifications of the percentage of ADM events in pancreatic acini depicted in Fig. 4d. Data are mean + s.d. of n=3 independent samples. ****p-value=10-15. h, qRT-PCRs validating YAPS127A overexpression in pancreatic acini shown in Fig. 4d. Data are mean + s.d. of n=3 independent samples. Data are normalized to 18-s rRNA. i, Pie chart of the main categories of GO entries associated to the HER2-induced genes that are dependent on YAP/TAZ and substrate stiffness, derived from the RNA-seq of Fig. 4e. Gene Ontology (GO) analysis was performed on the list of HER2-induced genes whose expression is dependent on YAP/TAZ and substrate stiffness (Supplementary Table 1). Of these genes, about 30% are linked to processes related to Cell Cycle progression, whereas others were classified as genes involved in Cell Fate Specification (10%), Signal Transduction (4%), RNA processing (22%) and Transcription (24%). P-values were calculated by unpaired two-sided Student’s t-test (d), one-way ANOVA with Sidak’s multiple comparisons test (c, e, f, h) and two-way ANOVA with Sidak’s multiple comparisons test (g).
Extended Data Fig. 5 Oncogenes modify the cell’s mechanical properties through Rac1 activation.
a, Western blots showing that the levels of YAP phosphorylation on the key LATS target residue S127 do not decrease after HER2-CA or KRasG12V expression in MCF10A cells. GAPDH serves as loading control. Phosphorylated vs. total YAP signal ratio (Empty: 1,19; HER2-CA: 1,24; KrasG12V: 0,93) was quantified by ImageJ analysis software. Results are representative of n=3 independent experiments, performed with similar results. b, Western blots showing that HER2-CA or KRasG12V expression in MCF10A cells does not affect the levels of the active pool of LATS kinases, as assessed by monitoring LATS1-activating phosphorylation on its key residue Thr1079. GAPDH serves as loading control. Phosphorylated vs. total LATS signal ratio (Empty: 1,28; HER2-CA: 1,12; KrasG12V: 1,11) was quantified by ImageJ analysis software. Results are representative of n=3 independent experiments, performed with similar results. c, Quantification of the colocalization of the cytoskeletal stainings for pMLC and F-actin shown in Fig. 5c. Data are as mean + s.d. of n=3 independent samples. ****p-value=2.42x10-6. d, Representative immunofluorescence images and quantifications showing that MCF10A cells transduced with Rac1-CA display increased formation of focal adhesion as visualized and quantified by colocalization of focal adhesion kinase (FAK) and Vinculin. Scale bars, 24 μm. Images and data are representative of n=3 independent experiments. Data are mean + s.d. ****p-value=6.27x10-4. e, Representative immunofluorescence images and quantifications showing that MCF10A cells transduced with Rac1-CA display increased maturation of focal adhesions as visualized and quantified by colocalization of phospho-focal adhesion kinase (pFAK) and Paxillin. Scale bars, 24 μm. Images and data are representative of n=3 independent experiments. Data are mean + s.d. *** p-value=2.41x10-4. f, Representative images and quantifications of collagen contraction assays performed with MCF10A cells transduced with Rac1-CA and treated with the indicated drugs. MCF10A cells transduced with empty vector are shown as negative control. Data are mean + s.d. of n>3 independent samples. ****p-value=4.51x10-6. g, Quantifications of the primary mammary colonies formed from Yap+/+;Taz+/+ or YapKO;TazKO mLD cells, treated as in Fig. 5g. Data are mean + s.d. of n= 3 independent experiments. h, Quantifications of the secondary and tertiary colonies derived from primary colonies formed by Rac1-CA-transduced mLD cells. Data are mean + s.d. of n= 3 independent experiments. i, Quantifications of colonies formed by hLD cells treated as in Fig. 5i. Data are mean + s.d. of n= 3 independent experiments. ****p-value=1.18x10-6. j, Luciferase assay in post-confluent HEK293 cells transfected with 8xGTIIC-Lux, and with empty vector or the indicated oncogene expressing vectors, alone or in combination with vectors coding for Rac1-DN. Data are mean + s.d. of n= 3 independent samples. k, qRT-PCRs of the ductal marker K19 in the pancreatic acini depicted in Fig 5l. Data are mean + s.d. of n=3 independent samples. l, qRT-PCRs showing effective YAPS127A overexpression and Rac1 depletion in pancreatic acini depicted in Fig. 5l. Data are presented as mean + s.d. of n=3 independent samples. m, Representative bright field images and quantifications of ADM events of pancreatic acini of the indicated genotypes treated with the Rac-specific GEFs Tiam1/Trio inhibitor NSC23766 (100 μM), or left untreated (Control). Images and data are representative of n=3 independent experiments. Scale bars, 70 μm. Data are mean + s.d. **** p-value=3.2x10-5. P-values were calculated by one-way ANOVA with Sidak’s multiple comparisons test (c–g, i, l, m) and two-way ANOVA with Sidak’s multiple comparisons test (j).
Extended Data Fig. 6 Transformation of immortalized cells requires sufficient substrate rigidity and YAP/TAZ activity.
a, Quantifications of colonies formed by control or oncogene-expressing MCF10A cells, cultured at the three indicated stiffnesses before embedding in agar. Data are mean + s.d. of n=3 independent samples. Controls showing the requirement of YAP/TAZ in similar cell transformation assays are shown in b. b, Quantifications of colonies formed by control or oncogene-expressing MCF10A cells, transfected with the indicated siRNAs before embedding in agar. YAP/TAZ depletion blunts oncogene-induced cell transformation. Data are mean + s.d. of n=3 independent samples. P-values were calculated by two-way ANOVA with Sidak’s multiple comparisons test.
Extended Data Fig. 7 Rheological measurements validating the purely elastic behavior of PAA hydrogels used in the study.
a, Rheological measurements showing Storage (G’) and Loss (G’’) moduli of PAA hydrogels used in the study as function of the frequency (from 0.01 to 1.259 Hz) in a constant-strain mode. Young’s modulus E=3G, is indicated above each panel. Results are representative of n=3 independent experiments, performed with similar results.
Supplementary information
Supplementary Information
Supplementary Fig. 1, Tables 1–3 and discussion
Source data
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Source Data Extended Data Fig. 4
Numerical data used to generate graphs in Extended Data Fig. 4
Source Data Extended Data Fig. 5
Numerical data used to generate graphs in Extended Data Fig. 5
Source Data Extended Data Fig. 6
Numerical data used to generate graphs in Extended Data Fig. 6
Source Data Extended Data Fig. 7
Numerical data used to generate graphs in Extended Data Fig. 7
Source Data Supplementary Table 1
List of GO terms significantly enriched (p-value ≤ 0.05) in HER2-induced genes whose expression is dependent on YAP/TAZ and substrate stiffness (referred to Extended Data Fig. 4i)
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Panciera, T., Citron, A., Di Biagio, D. et al. Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties. Nat. Mater. 19, 797–806 (2020). https://doi.org/10.1038/s41563-020-0615-x
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DOI: https://doi.org/10.1038/s41563-020-0615-x
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