SRC induces podoplanin expression to promote cell migration

Abstract

Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration.

FIGURE 1.

Layered culture system. a, nontransformed or Src-transformed Cx43Ko cells were incubated beneath or on top of a porous membrane that prevents actual mixing of the cell populations. Transformed cells separated by 1 mm from nontransformed cells enabled communication by diffusible factors but not intercellular junctions. Transformed cells incubated directly over nontransformed cells allowed for intercellular junctional communication and contact normalization. b, contact-normalized tumor cells were separately harvested and analyzed for v-Src or β-actin by Western blot analysis (20 μg/lane). c, each cell layer from the contact normalized configuration was separately harvested and analyzed by RT-PCR to detect mRNA encoding hygromycin phosphotransferase (Hygro; conferring hygromycin resistance to nontransformed cells), puromycin acetyltransferase (Puro; conferring puromycin resistance to transformed cells), or GAPDH (as a control). v-Src protein and puromycin acetyltransferase mRNA were retained in the transformed cell preparations, whereas hygromycin phosphotransferase mRNA was retained in the nontransformed cell preparations. Although RT-PCR data are only shown from nontransformed cells plated alone, all nontransformed cells in this system contained hygromycin phosphotransferase mRNA levels similar to nontransformed cells plated alone and no detectable puromycin acetyltransferase mRNA.

FIGURE 2.

RT-PCR analysis of the effects of Src and contact normalization on the expression of several genes. Src-transformed cells were cultured on porous membranes directly above nontransformed cells (contact normalized), directly above another layer of transformed cells (Src controls), or 1 mm above nontransformed cells (diffusible factors). Nontransformed and Src-transformed cells were also plated alone. Each cell layer was separately harvested and analyzed by RT-PCR to detect mRNA species as indicated. A control lane from reaction without reverse transcriptase is also indicated.

FIGURE 3.

Effects of Src and contact normalization on Pdpn, Tmem163, Fhl1, and GAPDH mRNA expression examined by microarrays. Src-transformed cells were cultured on porous membranes directly above nontransformed cells (contact normalized), directly above another layer of transformed cells (Src controls), or 1 mm above nontransformed cells (diffusible factors). Nontransformed and Src-transformed cells were also plated alone. Each cell layer was separately harvested and analyzed by Affymetrix expression microarrays to detect mRNA species as indicated. Data are shown as expression intensity from microarray analysis (mean ± S.E., n = 2). Configuration of cells in the layered culture system is presented on the top of the histograms, with the target cells filled in gray. Double and triple asterisks indicate p < 0.01 and p < 0.001 compared with Src-transformed cells by t test, respectively.

FIGURE 4.

qRT-PCR analysis of the effects of Src transformation and contact normalization on the expression of Pdpn and Fhl1 mRNA. mRNA transcripts encoding Pdpn and Fhl1 from nontransformed cells, Src-transformed cells, and contact-normalized transformed cells were examined by qRT-PCR. Values were standardized to GAPDH as an internal control and are shown as expression intensities relative to Src-transformed cells that were normalized to a value of 1.0 (mean ± S.E. (error bars), n = 4).

FIGURE 5.

Src utilizes Cas to augment Pdpn protein expression in transformed cells. a, protein from nontransformed and Src-transformed Cx43Ko cells, CasKo cells transfected with Cas or parental vectors, and wild type (WT) cells was analyzed by Western blotting to detect Pdpn, v-Src, active Src, Cas, and β-actin. b, Pdpn, Src, and nuclei were visualized by immunofluorescence microscopy in nontransformed and Src-transformed wild type and Cx43Ko cells. Differential interference contrast images are also shown. Scale bar, 100 μm.

FIGURE 6.

Pdpn expression is sufficient to increase migration of nontransformed cells. a, protein from nontransformed Cx43Ko cells transfected with cDNA encoding Pdpn, the empty parental vector (EF4), or v-Src was examined by Western blotting to detect Pdpn or β-actin. b, Pdpn (green) and nuclei (blue) were visualized by confocal immunofluorescence microscopy. Differential interference contrast images are also shown. Scale bar, 50 μm. c, cells (20,000/well) were cultured in anchored or nonanchored conditions and counted at the time points indicated. Data are shown as the number of cells/well (mean ± S.E. (error bars), with n = 2 for anchored growth and n = 4 for nonanchored growth). d, cells were plated on porous membranes and counted 24 h later. Migration was quantitated as the percent of cells that migrated from the top of the membrane to the bottom of the membrane, whereas colonization was quantitated as the percent of cells that migrated to the well beneath the membrane (mean ± S.E. (error bars), n = 3). Triple asterisks denote p < 0.001 compared with control transfectants by t test.

FIGURE 7.

Pdpn expression is required to promote migration of transformed cells. a, protein from Src-transformed Cx43Ko or wild type (WT) cells transfected with control siRNA or siRNA directed against Pdpn was examined by Western blotting to detect Pdpn or β-actin. b, cell migration was analyzed by wound healing and quantitated as the number of cells to enter a 1.8-mm² area in the middle of the wound in 24 h (mean ± S.E., n = 4). Triple asterisks denote p < 0.0001 compared with control transfectants by t test.