Yongquan Shen

Regulation of miRNA expression by Src and contact normalization: effects on nonanchored cell growth and migration

Transformation by the Src tyrosine kinase (Src) promotes nonanchored cell growth and migration. However, nontransformed cells can force Src-transformed cells to assume a normal morphology and phenotype by a process called ‘contact normalization’. It has become clear that microRNA (miRNA) can affect tumorigenesis by targeting gene products that direct cell growth and migration. However, the roles of miRNA in Src transformation or contact normalization have not yet been reported. We examined the expression of 95 miRNAs and found 9 of them significantly affected by Src. In this study, we report that miR-218 and miR-224 were most significantly induced by Src, but not affected by contact normalization. In contrast, miR-126 was most significantly suppressed by Src and was induced by contact normalization in transformed cells. Mir-126 targets Crk, a component of the focal adhesion network that participates in events required for tumor cell migration. Accordingly, we show that miR-126 expression correlates inversely with Crk levels, motility and the invasive potential of human mammary carcinoma cells. Moreover, we show that miR-224 expression promotes nonanchored growth of nontransformed cells. These data reveal novel insights into how Src regulates miRNA expression to promote hallmarks of tumor cell growth and invasion, and how nontransformed cells can affect miRNA expression in adjacent tumor cells to inhibit this process.

Src Uses Cas to Suppress Fhl1 in Order to Promote Nonanchored Growth and Migration of Tumor Cells

Anchorage independence and motility are hallmarks of tumor cell growth. Tumor cell growth and morphology can be normalized by contact with nontransformed cells. The Src tyrosine kinase phosphorylates specific sites on the focal adhesion adaptor protein Crk-associated substrate (Cas) to promote nonanchored cell growth and migration. We studied the effects of Src and Cas on the expression of >14,000 genes to identify molecular events that underlie these activities. Gene expression in tumor cells that were normalized by neighboring nontransformed cells was used as an additional filter to identify genes that control metastatic cell growth. This process enabled the identification of genes that play roles in anchorage-independent cell growth and migration. One candidate, four and a half LIM domains 1 (Fhl1), acts as a transcriptional regulator that can associate with cell junctions as well as with the nucleus. We show here that Src phosphorylates Cas to block Fhl1 expression. In addition, suppression of Fhl1 is required for Src to promote tumor cell growth. These data show that Fhl1 is a tumor suppressor gene that acts downstream of Src and Cas to specifically block anchorage-independent cell growth and migration. Moreover, Fhl1 was suppressed in tumors from several human tissues. Thus, identification of how Fhl1 controls fundamental aspects of tumor cell growth and metastasis may lead to the development of novel markers that can be used to diagnose human clinical specimens as well as open innovative avenues of investigations aimed at developing reagents that target cancer cells while minimizing damage to normal cells.

Src Induces Podoplanin Expression to Promote Cell Migration

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.

Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate.

The Src tyrosine kinase associates with the focal adhesion adaptor protein Cas (Crk‐associated substrate) to suppress the expression of potential tumor suppressor genes. For example, Src utilizes Cas to suppress the expression of the LIM‐only protein Fhl1 (four and a half LIM domains 1), in order to promote non‐anchored tumor‐cell growth and migration. Here, we report that the promoter region of the Fhl1 gene was methylated more in Src‐transformed cells than non‐transformed cells. In addition, global expression analysis indicates that Fhl1 induced expression of serum deprivation response factor (Sdpr) in Src‐transformed cells. Moreover, Fhl1 and Sdpr was expressed in approximately 87% and 40% of samples obtained from non‐transformed breast, 100% of samples obtained from non‐transformed kidney, and over 60% of samples obtained from non‐transformed prostate. In contrast, Fhl1 and Sdpr was detected in approximately 40% and 7% of matched samples from mammary carcinoma, less than 11% of matched samples from kidney carcinoma, and in less than 22% of matched samples from prostate carcinoma. These data indicate that Fhl1 and Sdpr expression was significantly reduced in tumors of the breast (P < 0.02 and P < 0.001), kidney (P < 0.01), and prostate (P < 0.05). In addition, although Src can activate mitogen‐activated protein kinase (MAPK) to promote tumor‐cell growth, our data indicate that Src did not rely on MAPK activity to suppress the expression of Fhl1 and Sdpr in transformed cells. Thus, Src induced methylation of the promoter region of the Fhl1 gene; Src suppressed Fhl1 and Sdpr expression independent of mitogen‐activated protein kinase (MAPK) activity; Fhl1 induced the expression of Sdpr in Src‐transformed cells; and Fhl1 and Sdpr expression was suppressed in tumors of the breast, kidney, and prostate. (Cancer Sci 2008; 99: 1326–1333)

Individual Cas Phosphorylation Sites Are Dispensable for Processive Phosphorylation by Src and Anchorage-independent Cell Growth

Cas is a multidomain signaling protein that resides in focal adhesions. Cas possesses a large central substrate domain containing 15 repeats of the sequence YXXP, which are phosphorylated by Src. The phosphorylation sites are essential for the roles of Cas in cell migration and in regulation of the actin cytoskeleton. We showed previously that Src catalyzes the multisite phosphorylation of Cas via a processive mechanism. In this study, we created mutant forms of Cas to identify the determinants for processive phosphorylation. Mutants containing single or multiple YXXP mutations were phosphorylated processively by Src, suggesting that individual sites are dispensable. The results also suggest that there is no defined order to the Cas phosphorylation events. We also studied the effects of these mutations by reintroducing Cas into Cas-deficient fibroblasts. Mutants lacking some or all YXXP sites augment the ability of Src to promote anchorage-independent growth. On the other hand, deletion of YXXP sites compromises the ability of Cas to promote tumor cell migration.

Plant Lectin Can Target Receptors Containing Sialic Acid, Exemplified by Podoplanin, to Inhibit Transformed Cell Growth and Migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Serines in the Intracellular Tail of Podoplanin (PDPN) Regulate Cell Motility

Background: PDPN is a transmembrane receptor that promotes cell migration, but modifications that regulate its effects are not known. Results: PKA can phosphorylate PDPN, nonphosphorylatable PDPN promotes cell migration, and phosphomimetic PDPN fails to promote cell migration. Conclusion: PKA can phosphorylate PDPN to decrease cell migration. Significance: PDPN effects on cell motility are important for processes including embryonic development and cancer progression. Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.

Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.

Abstract 4375: Podoplanin (PDPN): novel biomarker and chemotherapeutic target

Cancer is a leading cause of death. In fact, cancer killed over 8 million people around the world in 2012. Over 90% of these cancer deaths are due to metastasis, which results from tumor cell migration and invasion. Specific cancer biomarkers need to be identified in order to effectively target these motile cells. The transmembrane glycoprotein receptor podoplanin (PDPN) promotes tumor cell motility and metastasis in many aggressive cancers. Indeed, PDPN has emerged as a prime cancer biomarker and chemotherapeutic target. Here, we describe how PDPN can be targeted to inhibit the growth and motility of melanoma and oral cancer cells. PDPN has a short intracellular domain of 9 amino acids which include two conserved serine residues. PDPN also has a large extracellular domain that is extensively O-glycosylated with α2,3-sialic acid linked to galactose. We are developing novel methods to target the intracellular and extracellular domains of PDPN to combat cancer progression. For example, we have found that some activators of protein kinase A can induce phosphorylation of the intracellular serine residues of PDPN to inhibit tumor cell migration. In addition, we have found that Maackia amurensis seed lectin (MASL) can target the extracellular domain of PDPN to inhibit tumor cell growth, migration, and tumor progression in cell culture and animal models. Furthermore, we utilized live cell imaging to find that PDPN expression can be modulated in cancer associated fibroblasts to inhibit neighboring tumor cell migration and survival. Thus, reagents can be used to target PDPN from inside of the cell and outside of the cell to inhibit tumor cell migration and combat cancer progression. This work illuminates novel strategies designed to exploit PDPN as a functionally relevant biomarker and chemotherapeutic target. Citation Format: Harini Krishnan, Jhon Ochoa-alvarez, Yongquan Shen, Evan Nevel, David Kephart, Angels Nguyen, Min Han, Nimish Acharya, Robert Nagele, Maria Ramirez, W. Todd Miller, Evelyne Kalyoussef, Soly Baredes, Mahnaz Fatahzadeh, Lasse Jensen, Alan Shienbaum, Gary Goldberg. Podoplanin (PDPN): novel biomarker and chemotherapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2015-4375