Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression

Abstract

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers.

Figure 1. ECM stiffness modulates microRNA expression in culture and in vivo

A. Graphic depicting the in vitro experimental approach using human mammary epithelial cells (hMECs) and synthetic polyacrylamide (PA) substrates functionalized with recombinant basement membrane (BM) to mimic the mechanical properties of the normal human mammary gland and of mammary tumors during malignant progression. B. Graphic depicting the in vivo experimental approach inhibiting LOX using the polyoma middle T (PyMT) mouse model of breast cancer. C. Validation and quantification of miR-17-92 targets (with P<0.05 from the microarray results) with qPCR. miRNA targets were normalized with RNU48 and graphed relative to the soft group. D. qPCR of miR-18a for FVB mice (WT), FVB mice treated with a LOX inhibitor (WT+LOX-i), control PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i, n=10/group). Murine miR targets were normalized with U6 and graphed relative to the FVB (WT) group. E. Quantification of tumor growth and final tumor volume for PyMT primary tumor cells orthotopically injected into the #4 mammary gland of FVB hosts (n=6/group). PyMT cells expressed either a control construct (miR-CTL) or miR-18a. F. Quantification of PyMT mRNA expression from the lungs of host FVB mice orthotopically injected with PyMT cells expressing either a control construct (miR-CTL) or miR-18a (n=6/group). Results were normalized to 18S and graphed relative to miR-CTL. G. Quantification of tumor growth and final tumor volume for PyMT primary tumor cells orthotopically injected into the #4 mammary gland of FVB hosts (n=5/group). PyMT cells expressed either a control antagomiR (ant-CTL) or an antagomiR to miR-18a (ant-18a). H. Quantification of lung weight for FVB mice tail-vein injected with PyMT primary cells expressing either a control antagomiR (ant-CTL) or an antagomiR to miR-18a (ant-18a, n=6/group). For in vitro bar graphs, results are the mean ± S.E.M. of at least 3 independent experiments. For in vivo bar graphs, results are the mean ± S.D. (*, P<0.05; **, P<0.01; ***, P< 0.001)

Figure 2. ECM stiffness promotes malignancy by inducing miR-18a to reduce PTEN and enhance PI3K activity

A. Luciferase reporter analysis of either wild-type or mutated PTEN 3′UTR activity (for two putative miRNA seed regions) upon addition of 1μg of either a control (miR-CTL) or miR-18a. Results are normalized to respective miR-CTL groups. B. PTEN mRNA for hMECs cultured on soft (<400Pa) or stiff (>5kPa) PA gels. Results are normalized to 18S and graphed relative to soft. C. Immunofluorescence images for PTEN (red) and DAPI (blue) in hMECs cultured on soft or stiff PA gels. Scale bar, 20μm; inset scale bar, 50μm. D. Nuclear and cytoplasmic PTEN protein for hMECs cultured on soft or stiff PA gels. Results are normalized to pHistone H3 (nuclear) or Gapdh (cytoplasmic), and graphed relative to soft. E. PTEN mRNA for FVB mice (WT), FVB mice treated with a LOX inhibitor (WT+LOX-i), control PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Results are normalized to 18S and graphed relative to WT (n=10/group). F. Immunofluorescent images of Pten (top, red), pS473-Akt (bottom, red) and DAPI (blue) of mammary glands from FVB mice (WT), FVB mice treated with a LOX inhibitor (WT+LOX-i), malignant PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Scale bar, 20μm; inset scale bar, 50μm. G. PTEN protein expression for hMECs cultured on soft or stiff substrates expressing a microRNA antagomiR control (ant-CTL), an antagomiR to miR-18a (ant-18a) or an antagomiR to miR-19b (ant-19b). Results are normalized to β-actin and graphed relative to soft vector. H. PTEN protein expression for hMECs cultured on soft substrates expressing the control vector (miR-CTL), miR-18a, or without expression vectors (hMECs). Results are normalized to β-actin and graphed relative to hMECs. I. Pten mRNA expression for orthotopic PyMT tumors expressing either a microRNA antagomiR control (ant-CTL) or an antagomiR to miR-18a (ant-18a, n=5/group). Results are normalized to 18S and graphed relative to ant-CTL. J. Pten mRNA expression for orthotopic PyMT tumors expressing either a microRNA control vector (miR-CTL) or miR-18a (n=6/group). Results are normalized to 18S and graphed relative to miR-CTL. In all in vitro bar graphs, results are the mean ± S.E.M. of at least 3 independent experiments. For in vivo bar graphs, results are the mean ± S.D. (*, P<0.05; **, P<0.01; ***, P< 0.001)

Figure 3. ECM stiffness promotes malignancy by inducing miR-18a to reduce HOXA9

A. Luciferase reporter analysis of either wild-type or mutated HOXA9 3′UTR activity upon addition of 1μg of either a control vector (miR-CTL) or miR-18a. Results are normalized to respective miR-CTL groups. B. HOXA9 protein for hMECs cultured on soft (<400Pa) or stiff (>5kPa) PA gels. Results are normalized to Gapdh and graphed relative to soft. C. HOXA9 mRNA for hMECs cultured on soft or stiff PA gels. Results are normalized to Gapdh and graphed relative to SOFT. D. Immunofluorescence images for HOXA9 (red), PTEN (green) and DAPI (blue) in hMECs cultured on soft or stiff PA gels. Scale bar, 20μm; inset scale bar, 50μm. E. HOXA9 mRNA for FVB mice (WT), control PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Results are normalized to 18S and graphed relative to WT (n=10/group). F. Immunofluorescence images of HOXA9 (red) and DAPI (blue) for FVB mice (WT), FVB mice treated with a LOX inhibitor (WT+LOX-i), control PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Scale bar, 20μm; inset scale bar, 50μm. G. HOXA9 protein for hMECs cultured on soft or stiff substrates expressing an microRNA antagomiR control, an antagomiR to miR-18a (ant-18a) or an antagomiR to miR-19b (ant-19b). Results are normalized to β-actin and graphed relative to soft vector. H. HOXA9 protein for hMECs cultured on soft substrates expressing the control vector (miR-CTL), miR-18a, or without expression vectors (hMECs). Results are normalized to β-actin and graphed relative to hMECs. I. Hoxa9 mRNA expression for orthotopic PyMT tumors expressing either a microRNA antagomiR control (ant-CTL) or an antagomiR to miR-18a (ant-18a, n=5/group). Results are normalized to 18S and graphed relative to ant-CTL. J. Hoxa9 mRNA expression for orthotopic PyMT tumors expressing either a microRNA control vector (miR-CTL) or miR-18a (n=6/group). Results are normalized to 18S and graphed relative to miR-CTL. In all in vitro bar graphs, results are the mean ± S.E.M. of at least 3 independent experiments. For in vivo bar graphs, results are the mean ± S.D. (*, P<0.05; **, P<0.01; ***, P< 0.001)

Figure 4. ECM stiffness promotes malignancy by preventing HOXA9-dependent PTEN transcription

A. PTEN mRNA expression in hMECs expressing a control shRNA (Vector) or an shRNA to HOXA9 (HOXA9-i), and cultured on a soft (<400Pa) substrate. Results are normalized to 18S and graphed relative to Vector. B. PTEN mRNA expression in hMECs cultured on a soft (<400Pa) or stiff (>5kPa) substrate, ectopically expressing a control vector (Vector) or HOXA9. Results are normalized to 18S and graphed relative to soft. C. PTEN mRNA expression in malignant hMECs (T4-2 or MDA-MB-231 cells) ectopically expressing a control vector (Vector) or HOXA9. Results are normalized to 18S and graphed relative to soft. D. Quantification of tumor growth and final tumor volume for PyMT primary tumor cells expressing either a control vector (Vector) or Hoxa9, and orthotopically injected into the #4 mammary gland of FVB hosts (n=5/group). E. miR-18a expression for orthotopic PyMT tumors expressing either a control vector (Vector) or Hoxa9 (n=5/group). Results are normalized to 18S and graphed relative to Vector. F. Pten and Hoxa9 expressions for orthotopic PyMT tumors expressing either a control vector (Vector) or Hoxa9 (n=5/group). Results are normalized to 18S and graphed relative to Vector. G. Luciferase reporter analysis of PTEN promoter activity in response to the addition of wild-type HOXA9. H. Luciferase reporter analysis of PTEN promoter activity upon addition of 2μg HOXA9 containing an N255T (DNA BM) mutation in the conserved DNA binding domain or the addition of 2μg of HOXA10. I. Luciferase reporter analysis of PTEN promoter activity in response to 1μg HOXA9 with 2μg of either PBX1 or MEIS1a. J. Representative gel of ChIP studies in hMECs, revealing co-precipitation of HOXA9 with the PTEN promoter. Quantification of the chromatin immunoprecipitation results of HOXA9 on the PTEN proximal promoter for non-malignant hMECs expressing either a shRNA control (Vector) or an shRNA to HOXA9 (HOXA9-i). K. Graphic depicting a model for suppression of PTEN directly via miR-18a and indirectly through miR-18a regulation of HOXA9. In all in vitro bar graphs, results are the mean ± S.E.M. of at least 3 independent experiments. For in vivo bar graphs, results are the mean ± S.D. (*, P<0.05; **, P<0.01; ***, P< 0.001)

Figure 5. Tissue stiffness engages mechanotransduction signaling pathways to promote miR-18a dependent malignancy

A. MYC mRNA and protein expressions in hMECs cultured on PA gels of increasing stiffness. Results are normalized to GAPDH and graphed relative to soft (<400Pa). B. MYC mRNA expression in malignant T4-2 hMECs cultured on soft (<400Pa) and stiff (>5kPa) PA gels. Results are normalized to GAPDH and graphed relative to soft. C. miR-18a expression for hMECs cultured on a soft substrate (<400Pa), or on a stiff substrate (>5kPa) with 10μM of the MYC inhibitor 10058-F4 (MYC-i). Results are normalized to RNU48 and graphed relative to soft. D. Left: Myc mRNA expression in mammary glands from FVB mice (WT), FVB mice treated with a LOX inhibitor (WT+LOX-i), malignant PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Results are normalized to 18S and graphed relative to WT (n=10/group). Right: Myc protein expression in mammary glands from malignant PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Results are graphed relative to Tumor (n=5/group). E. Left: Active β-catenin protein expression normalized to total β-catenin, in hMECs cultured on PA gels of increasing stiffness. Results are normalized to total β-catenin and graphed relative to soft. Right: Immunofluorescence images of β-catenin (red) and DAPI (blue) in hMECs cultured on PA gels of increasing stiffness. Scale bar, 50μm; inset scale bar, 5μm. F. Active β-catenin protein expression normalized to total β-catenin in mammary glands from malignant PyMT mice (Tumor) and PyMT mice treated with a LOX inhibitor (LOX-i). Results are graphed relative to Tumor (n=5/group). G. pFAK³⁹⁷ protein expression in hMECs cultured on PA gels of increasing stiffness. Results are normalized to GAPDH and graphed relative to soft. H. miR-18a expression for hMECs cultured on a stiff substrate (>5kPa) with and without 1μM of the FAK inhibitor, FAK inhibitor 14 (FAK-i). Results are normalized to RNU48 and graphed relative to CTL (no inhibitor). I. HOXA9 and PTEN protein expressions for hMECs cultured on a stiff substrate (>5kPa) with and without 1μM of FAK inhibitor 14 (FAK-i). Results are normalized to GAPDH and graphed relative to CTL (no inhibitor). J. Immunofluorescence images of pFAK^Y397 (red) and DAPI (blue) in FVB (WT) and transgenic V737N mice (n=6/group). Scale bar, 20μm. K. β-catenin nuclear expression in FVB (WT) and transgenic V737N mice (n=6/group). Results are normalized to total β-catenin expression and graphed relative to WT. Scale bar, 20μm; inset scale bar, 5μm. L. Myc mRNA and miR-18a expressions in FVB (WT) and transgenic V737N mice (n=6/group). Results are normalized to 18S and sno202 respectively, and graphed relative to WT. M. Pten and Hoxa9 protein expressions in FVB (WT) and transgenic V737N mice (n=6/group). Scale bar, 20μm; inset scale bar, 5μm. In all in vitro bar graphs, results are the mean ± S.E.M. of at least 3 independent experiments. For in vivo bar graphs, results are the mean ± S.D. (*, P<0.05; **, P<0.01; ***, P< 0.001)

Figure 6. Breast malignancy associates with increased miR-18a and reduced PTEN expression

A. miR-18a in human non-malignant breast samples (Normal) and breast tumor samples (luminal A, luminal B, basal-like and HER2⁺). Results are normalized to RNU48 and graphed relative to Normal. B. Correlation between miR-18a expression and elastic modulus (upper quartile) in human patient samples (both non-malignant and tumor samples combined). C. Elastic moduli (upper quartile) for luminal A and luminal B breast tumor samples. D. PTEN (left) and HOXA9 (right) mRNA expressions in human non-malignant breast (Normal) samples and breast tumor samples (luminal A, luminal B, basal-like and HER2+). Results are normalized to 18S and graphed relative to Normal. E. Correlation between HOXA9 and PTEN mRNA levels in human patient samples (both non-malignant and tumor samples combined). F. HOXA9 (top, red), PTEN (middle, red), pAKT substrate (bottom, red) and DAPI (blue) for human non-malignant breast samples (Normal) and breast tumor samples (luminal A, luminal B, basal-like and HER2+). Scale bar, 100μm. G. Kaplan-Meier graph (left) showing that patients with luminal breast cancers whose tumors expressed the highest miR-18a levels (highest expression quartile; red line) experienced significantly reduced metastasis-free survival compared with patients in the lowest quartile (black line). Kaplan-Meier graph (right) showing patients with basal-like and HER2+ breast cancers whose tumors expressed the highest miR-18a levels (highest expression quartile; red line) and lowest miR-18a levels (black line). H. Graphic depicting a model for suppression of PTEN directly via β-catenin stimulation of MYC-driven miR-18a and indirectly through miR-18a regulation of HOXA9. (*, P<0.05; **, P<0.01; ***, P< 0.001)