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Integrin α3β1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome.

ABSTRACT: The development of integrin-targeted cancer therapies is hindered by incomplete understanding of integrin function in tumor cells and the tumor microenvironment. Previous studies showed that mice with epidermis-specific deletion of the α3 integrin subunit fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for integrin α3β1. Here, we generated mice with tamoxifen-inducible, epidermis-specific α3 knockout to determine the role of α3β1 in the maintenance of established tumor cells and/or the associated stroma. Genetic ablation of α3 in established skin tumors caused their rapid regression, indicating that α3β1 is essential to maintain tumor growth. Although reduced proliferation and increased apoptosis were observed in α3β1-deficient tumor cells, these changes followed a robust increase in stromal apoptosis. Furthermore, macrophages and fibulin-2 levels were reduced in stroma following α3 deletion from tumor cells. Mass spectrometric analysis of conditioned medium from immortalized keratinocytes showed that α3β1 regulates a substantial fraction of the keratinocyte secretome, including fibulin-2 and macrophage CSF1; RNA in situ hybridization showed that expression of these two genes was reduced in tumor keratinocytes in vivo. Our findings identify α3β1 as a regulator of the keratinocyte secretome and skin tumor microenvironment and as a potential therapeutic target.

SUBMITTER: Longmate WM

PROVIDER: S-EPMC7680721 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Integrin α3β1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome.

Longmate Whitney M WM Varney Scott S Power Derek D Miskin Rakshitha Pandulal RP Anderson Karl E KE DeFreest Lori L Van De Water Livingston L DiPersio C Michael CM

The Journal of investigative dermatology 20200523 1

The development of integrin-targeted cancer therapies is hindered by incomplete understanding of integrin function in tumor cells and the tumor microenvironment. Previous studies showed that mice with epidermis-specific deletion of the α3 integrin subunit fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for integrin α3β1. Here, we generated mice with tamoxifen-inducible, epidermis-specific α3 knockout to determine the role of α3β1 in the maintenan ...[more]

PMID: 32454065

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Project description:Integrin α3β1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3β1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3β1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3β1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3β1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3β1-expressing keratinocytes compared with α3β1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3β1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3β1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3β1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in α3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in α3β1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin α3β1-mediated gene regulation through alternative polyadenylation.

Project description:BackgroundHER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3β1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3β1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis.MethodsTo investigate the role of α3β1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3β1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3β1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence.ResultsTumor onset in mice was independent of the presence of α3β1. In contrast, the depletion of α3β1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3β1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow.ConclusionDownregulation of α3β1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3β1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3β1 in breast cancer.

Dataset Information

Integrin α3β1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome.

Publications

Integrin α3β1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome.

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