Elsevier

Matrix Biology

Volume 87, May 2020, Pages 26-47
Matrix Biology

H-Ras activation and fibroblast-induced TGF-β signaling promote laminin-332 accumulation and invasion in cutaneous squamous cell carcinoma

https://doi.org/10.1016/j.matbio.2019.09.001Get rights and content

Highlights

  • ECM component laminin-332 is produced by cancer cells located in the invasive front of cSCC tumor samples.

  • TGF-β signaling promotes laminin-332 synthesis in an active H-Ras-dependent manner, which increases cancer cell invasion.

  • Ras and TGF-β signaling pathways are coactivated in vivo in conjunction with laminin-332 accumulation.

  • We unveil a molecular mechanism that explains the high level of laminin-332 in human cSCC tumors.

  • We provide compelling evidence that fibroblasts have an integral role in the progression of metastatic cSCC.

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.

Introduction

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide [1,2]. cSCC progresses from premalignant lesion, actinic keratosis, to carcinoma in situ (cSCCIS) and finally to invasive and metastatic disease [2]. The molecular basis of cSCC progression is incompletely understood, but activating mutations in HRAS, KRAS and epidermal growth factor receptor (EGFR) have been found [3,4]. Previously, it has been suggested that PI3K signaling pathway participates in the cSCC progression. Ras signaling alone, however, was not found to be sufficient to activate PI3K/AKT pathway in cSCC tumors, and therefore it was speculated that signals from the tumor stroma may also be needed for PI3K activation and subsequent cSCC tumorigenesis [5].

Tumor stroma, or tumor microenvironment, is a complex meshwork of extracellular matrix (ECM), activated fibroblasts, immune cells and capillary vessels [6]. Currently, indisputable evidence show that cancer progression is not led solely by cancer cells but that the tumor microenvironment plays a critical role in the development of aberrant tissue functions and malignancies [[7], [8], [9]]. In this microenvironment, fibroblasts are the most abundant cell type, and by producing soluble proteins, such as growth factors and various ECM components they control the behaviour of other cell types. Recently, numerous studies have shown that fibroblasts have a significant functional role in nearly all aspects of tumor progression. They promote tumor growth and invasion, induce angiogenesis, modulate inflammation and promote chemoresistance [[10], [11], [12]].

In the past few years, three-dimensional (3D) cell cultures have gained increasing interest while the limitations of traditional two-dimensional (2D) monolayer cell cultures have been recognized. The cells in monolayer cultures cannot form the same natural organization as the cells in vivo in a tissue, in which all cells are surrounded by other cells or the ECM. Spheroids provide more tissue-like environment, which enables the cellular interactions that are vital for the natural function of the cells. In addition to close cell–cell/cell–ECM interactions, spheroids of cancer cells also limit oxygen and nutrition flow into the cells resulting in different populations of proliferating, quiescent and necrotic cells [13]. Because of these features cancer cell spheroids resemble solid tumors and can serve as an in vitro model to better mimic the in vivo tumor tissue properties.

In the present study, we show how fibroblasts modify ECM expression in tumor-like 3D spheroids and how they influence on cancer cell invasion. First, we observed that in cSCC tumor samples laminin-332 is frequently produced by cancer cells located in the invasive front. To study this further we used HaCaT/ras-HaCaT human keratinocyte carcinogenesis model which mimics the progression of cSCC [14]. Our data indicate that when cocultured together with fibroblasts, the transformed epithelial cells show increased invasive capacity and they also produce high levels of laminin-332. The invasion and the increase in laminin-332 production are shown to require concomitant activation of H-Ras and TGF-β signaling pathways in cancer cells. These results unveil a molecular mechanism that explains the high level of laminin-332 in human cSCC tumors, which in turn promotes cancer cell invasion.

Section snippets

Laminin-332 γ2 chain is accumulated in the invasive edge in cSCC tumors

Immunohistochemical analyses have revealed high laminin-332 γ2 chain accumulation in cSCC, and also its increased production in pre-malignant conditions, such as actinic keratosis and cSCCIS [15,16]. To investigate the localization of laminin-332 γ2 chain in more detail, we analyzed with immunohistochemistry (IHC) a large panel of sporadic UV-induced invasive cSCCs (n = 208) in tissue microarrays (TMA). Prominent tumor cell-specific immunostaining of laminin γ2 was noted in cSCC tumor cell

Discussion

Laminin-332 is a major protein component in skin basement membrane (BM). Basal keratinocytes use integrin type adhesion receptors, namely α6β4 and α3β1 heterodimers, in anchorage to this laminin in the lamina rara layer of BM [17]. Furthermore, laminin-332 also binds to anchoring fibrils formed by collagen VII [40], and it is also found deeper in the dermis in the anchoring plaques [41]. In cSCCs normal BM structures disappear, potentially due to the increased proteolytic activity. Importantly,

Ethical issues

Approval for the use of archival tissue specimens of primary cSCCs was obtained from the Ethics Committee of the Hospital District of the Southwest Finland, Turku, Finland. The study was performed in accordance with the Declaration of Helsinki Principles and with the approval of Turku University Hospital. Written informed consent of all participants was obtained before surgery. All the methods used in this study were carried out in accordance with the relevant guidelines and regulations. The

Author contributions

J.H. and V-M.K. conceived the project; J.H., V-M.K. and E.S. planned the experiments; E.S. performed the cell experiments, confocal imaging and data analysis; P.R. conducted mass spectrometry experiments and data analysis; P. Riihilä and L.N. planned and generated TMA blocks and planned IHC experiments and analysis. P. Riihilä performed immunohistochemical experiments and data analysis; E.S., J.H. and V-M.K. wrote the manuscript. All authors contributed to manuscript preparation and approved

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [80] partner repository with the dataset identifier PXD013113. All other data in this article are available by contacting the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Acknowledgements

This research was supported by the grants from Jane and Aatos Erkko Foundation, Sigrid Jusélius Foundation, Finnish Cancer Research Foundation and Turku University Hospital (project 13336). Mass spectrometry analyses were performed at the Turku Proteomics Facility, supported by Biocenter Finland. We thank the Core Facilities of the Institute of Biomedicine in University of Turku for immunohistochemistry stainings and Auria Biobank in Turku University Hospital and the University of Turku for TMA

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