Heterocellular N-cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells

Abstract

Background The Src tyrosine kinase phosphorylates effector proteins to induce expression of the podoplanin (PDPN) receptor in order to promote tumor progression. However, nontransformed cells can normalize the growth and morphology of neighboring transformed cells. Transformed cells must escape this process, called “contact normalization”, to become invasive and malignant. Contact normalization requires junctional communication between transformed and nontransformed cells. However, specific junctions that mediate this process have not been defined. This study aimed to identify junctional proteins required for contact normalization. Methods Src transformed cells and oral squamous cell carcinoma cells were cultured with nontransformed cells. Formation of heterocellular adherens junctions between transformed and nontransformed cells was visualized by fluorescent microscopy. CRISPR technology was used to produce cadherin deficient and cadherin competent nontransformed cells to determine the requirement for adherens junctions during contact normalization. Contact normalization of transformed cells cultured with cadherin deficient or cadherin competent nontransformed cells was analyzed by growth assays, immunofluorescence, western blotting, and RNA-seq. In addition, Src transformed cells expressing PDPN under a constitutively active exogenous promoter were used to examine the ability of PDPN to override contact normalization. Results We found that N-cadherin (N-Cdh) appeared to mediate contact normalization. Cadherin competent cells that expressed N-Cdh inhibited the growth of neighboring transformed cells in culture, while cadherin deficient cells failed to inhibit the growth of these cells. Results from RNA-seq analysis indicate that about 10% of the transcripts affected by contact normalization relied on cadherin mediated communication, and this set of genes includes PDPN. In contrast, cadherin deficient cells failed to inhibit PDPN expression or normalize the growth of adjacent transformed cells. These data indicate that nontransformed cells formed heterocellular cadherin junctions to inhibit PDPN expression in adjacent transformed cells. Moreover, we found that PDPN enabled transformed cells to override the effects of contact normalization in the face of continued N-Cdh expression. Cadherin competent cells failed to normalize the growth of transformed cells expressing PDPN under a constitutively active exogenous promoter. Conclusions Nontransformed cells form cadherin junctions with adjacent transformed cells to decrease PDPN expression in order to inhibit tumor cell proliferation. Plain English Summary Cancer begins when a single cell acquires changes that enables them to form tumors. During these beginning stages of cancer development, normal cells surround and directly contact the cancer cell to prevent tumor formation and inhibit cancer progression. This process is called contact normalization. Cancer cells must break free from contact normalization to progress into a malignant cancer. Contact normalization is a widespread and powerful process; however, not much is known about the mechanisms involved in this process. This work identifies proteins required to form contacts between normal cells and cancer cells, and explores pathways by which cancer cells override contact normalization to progress into malignant cancers. Graphical abstract

Independent effects of Src kinase and podoplanin on anchorage independent cell growth and migration

“…[26][27][28] Src kinase activity causes a pronounced effect on cell morphology. 4,13,18,29,30 Cells expressing tsSrc assumed a disorganized and refractive morphology typical of transformed cells at the permissive temperature, while they assumed a flattened morphology in contact inhibited monolayers typical of nontransformed cells at the nonpermissive temperature. These changes occurred regardless of PDPN expression, indicating that Src does not require PDPN to disrupt cell shape.…”

“…To examine actin fiber morphology, cells were cultured on 35 mm poly‐ d ‐lysine glass bottom dishes (MatTek Corp, P35GC‐1.5‐14‐C) at the nonpermissive (39°C) or permissive (34°C) temperature for 72 h, fixed with 4% paraformaldehyde, permeabilized with 1% Triton X‐100, and then stained with phalloidin‐Texas Red (Invitrogen, T7471) and Hoechst (Molecular Probes, 33342) according to manufacturer's recommendations. Cells were visualized on a Carl Zeiss Axio Observer Z1 equipped with a Plan‐Apochromat ×63 objective, apotome 2, filter sets to detect RFP (excitation 560 ± 40, emission 630 ± 75) and Hoechst (excitation 390 ± 22, emission 460 ± 50) with a Zeiss AxioCam Mrc camera Rev3 equipped with Zen Pro 2.3 software as previously described 15,17,18 …”

“…Cells were visualized on a Carl Zeiss Axio Observer Z1 equipped with a Plan-Apochromat ×63 objective, apotome 2, filter sets to detect RFP (excitation 560 ± 40, emission 630 ± 75) and Hoechst (excitation 390 ± 22, emission 460 ± 50) with a Zeiss AxioCam Mrc camera Rev3 equipped with Zen Pro 2.3 software as previously described. 15,17,18…”