Seo Hee Nam

Interaction of tetraspan(in) TM4SF5 with CD44 promotes self‐renewal and circulating capacities of hepatocarcinoma cells

Tumor metastasis involves circulating and tumor‐initiating capacities of metastatic cancer cells. Epithelial‐mesenchymal transition (EMT) is related to self‐renewal capacity and circulating tumor cell (CTC) characteristics for tumor metastasis. Although tumor metastasis is a life‐threatening, complicated process that occurs through circulation of tumor cells, mechanistic aspects of self‐renewal and circulating capacities have been largely unknown. Hepatic transmembrane 4 L six family member 5 (TM4SF5) promotes EMT for malignant growth and migration, so it was rationalized that TM4SF5, as a hepatocellular carcinoma (HCC) biomarker, might be important for metastatic potential. Here, self‐renewal capacity by TM4SF5 was mechanistically explored using hepatocarcinoma cells with or without TM4SF5 expression, and we explored whether they became CTCs using mouse liver‐orthotopic model systems. We found that TM4SF5‐dependent sphere growth correlated with CD24−, aldehyde dehydrogenase (ALDH) activity, as well as a physical association between CD44 and TM4SF5. Interaction between TM4SF5 and CD44 was through their extracellular domains with N‐glycosylation modifications. TM4SF5/CD44 interaction activated proto‐oncogene tyrosine‐protein kinase Src (c‐Src)/signal transducer and activator of transcription 3 (STAT3)/Twist‐related protein 1 (Twist1)/B‐cell‐specific Moloney murine leukemia virus integration site 1 (Bmi1) signaling for spheroid formation, whereas disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts using 200∼5,000 cells per injection, TM4SF5‐positive tumors exhibited subpopulations with locally increased CD44 expressions, supporting for tumor cell differentiation. TM4SF5‐positive, but not TM4SF5‐ or CD44‐knocked‐down, cells were identified circulating in blood 4‐6 weeks after orthotopic liver injection using in vivo laser scanning endomicroscopy. Anti‐TM4SF5 reagent blocked their metastasis to distal intestinal organs. Conclusion: TM4SF5 promotes self‐renewal and CTC properties supported by TM4SF5+/CD44+(TM4SF5‐bound)/ALDH+/CD24− markers during HCC metastasis. (Hepatology 2015;61:1978‐1997)

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

Correlations between transmembrane 4 L6 family member 5 (TM4SF5), CD151, and CD63 in liver fibrotic phenotypes and hepatic migration and invasive capacities.

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl4-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.

Cross Talk between the TM4SF5/Focal Adhesion Kinase and the Interleukin-6/STAT3 Pathways Promotes Immune Escape of Human Liver Cancer Cells

ABSTRACT TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway.

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

Bidirectional signaling between TM4SF5 and IGF1R promotes resistance to EGFR kinase inhibitors

OBJECTIVES The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial-mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). MATERIALS AND METHODS Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. RESULTS AND CONCLUSION We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC.

Differential regulation of cellular functions by the C-termini of transmembrane 4 L six family proteins in 2- or 3-dimensional environment

The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus.

Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52 phosphorylation and the nucleus after S207 phosphorylation), leading to probably alternative noncanonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within 3D gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous KRS and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in 3D gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GRO&agr;/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminin production. Analyses of tissues from clinical colon cancer patients and Krs–/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.

CD133-induced TM4SF5 expression promotes sphere growth via recruitment and blocking of protein tyrosine phosphatase receptor type F (PTPRF)

CD133 is a surface marker of liver cancer stem cells. Transmembrane 4 L six family member 5 (TM4SF5) promotes sphere growth and circulation. However, it is unknown how CD133 and TM4SF5 cross-talk with each other for cancer stem cell properties. Here, we investigated the significance of inter-relationships between CD133, TM4SF5, CD44, and protein tyrosine phosphatase receptor type F (PTPRF) in a three-dimensional (3D) sphere growth system. We found that CD133 upregulated TM4SF5 and CD44, whereas TM4SF5 and CD44 did not affect CD133 expression. Signaling activity following CD133 phosphorylation caused TM4SF5 expression and sphere growth. TM4SF5 bound to CD133 and promoted c-Src activity for CD133 phosphorylation as a positive feedback loop, leading to CD133-mediated sphere growth that was inhibited by TM4SF5 inhibition or suppression. TM4SF5 also bound PTPRF and promoted paxillin phosphorylation. Decreased sphere growth upon CD133 suppression was recovered by TM4SF5 expression and partially by PTPRF suppression. TM4SF5 inhibition enhanced PTPRF levels and abolished PTPRF suppression-mediated sphere growth. Altogether, CD133-induced TM4SF5 expression and function were important for liver cancer sphere growth and may be a promising target to block metastasis.

TM4SF5 promotes metastatic behavior of cells in 3D extracellular matrix gels by reducing dependency on environmental cues.

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.