Jun Chung

Exosomes in Cancer Diagnostics

Exosomes are endosome derived extracellular vesicles of 30–120 nm size ranges. Exosomes have been identified as mediators of cell-to-cell communication by transferring bioactive molecules such as nucleic acids, proteins and lipids into recipient cells. While exosomes are secreted by multiple cell types, cancer derived exosomes not only influence the invasive potentials of proximally located cells, but also affect distantly located tissues. Based on their ability to alter tumor microenvironment by regulating immunity, angiogenesis and metastasis, there has been growing interest in defining the clinical relevance of exosomes in cancers. In particular, exosomes are valuable sources for biomarkers due to selective cargo loading and resemblance to their parental cells. In this review, we summarize the recent findings to utilize exosomes as cancer biomarkers for early detection, diagnosis and therapy selection.

Integrin (α6β4) Signals Through Src to Increase Expression of S100A4, a Metastasis-Promoting Factor: Implications for Cancer Cell Invasion

Integrin α6β4 is linked to cancer cell motility and invasion in aggressive and metastatic cancer cells. In this study, we showed that expression of the β4 integrin in MDA-MB-435 cancer cells (MDA-MB-435/β4) leads to a dramatic increase in expression of a metastasis-promoting factor, S100A4, as determined by affymetrix gene chip microarray, quantitative real-time PCR, and Western blot analysis. Alternatively, knocking down β4 integrin expression in MDA-MB-231 breast carcinoma cells by shRNA reduced the level of S100A4 expression. The mechanism by which α6β4 enhances S100A4 expression involves Src, Akt, and NFAT. We have further shown that Y1494, a tyrosine residue of the ITIM motif in the cytoplasmic domain of the β4 integrin subunit, is essential for α6β4-dependent S100A4 expression. Reduction of S100A4 expression by shRNA blocked migration, invasion, and anchorage-independent growth of MDA-MB-435/β4, SUM-159, and MDA-MB-231 cells. These studies define a novel mechanism by which integrin α6β4 promotes cancer cell motility and invasion, and provides insight into how S100A4 expression is regulated in cancer cells. (Mol Cancer Res 2009;7(10):1605–12)

Role of α6β4 Integrin in Cell Motility, Invasion and Metastasis of Mammary Tumors

Integrin α6β4 is the receptor for the laminin family of extracellular matrix proteins and is widely expressed in most epithelial tissues and Schwann cells. The expression of this integrin is up-regulated in most epithelial tumors, suggesting the role of α6β4 in their progression. The tumor microenvironment is also known to enhance the signaling competence of α6β4 through functional and physical interactions with other receptors. In this review, we discuss the biological mechanisms by which integrin α6β4 promotes carcinoma cell motility and invasion that leads to mammary tumor progression.

Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells

Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

Epigenetic silencing of ARRDC3 expression in basal-like breast cancer cells

Arrestin domain-containing 3 (ARRDC3) is a tumor suppressor whose expression is either lost or suppressed in basal-like breast cancer (BLBC). However, the mechanism by which BLBC suppresses ARRDC3 expression is not established. Here, we show that expression of ARRDC3 in BLBC cells is suppressed at the transcriptional level. Suppression of ARRDC3 expression in BLBC cells involves epigenetic silencing as inhibitors of class III histone deacetylases (HDACs) significantly restores ARRDC3 levels in BLBC cells. SIRT2, among class III HDACs, plays a major role in epigenetic silencing of ARRDC3 in MDA-MB-231 cells. Acetylation levels of the ARRDC3 promoter in BLBC cells is significantly lower than that of other sub-types of BC cells. Chromatin immunopreciptitation analysis established SIRT2 binding at ARRDC3 promoter in BLBC cells. Our studies indicate that SIRT2 dependent epigenetic silencing of ARRDC3 is one of the important events that may contribute to the aggressive nature of BLBC cells.

The role of c-Src in integrin (α6β4) dependent translational control

BackgroundIntegrin α6β4 contributes to cancer progression by stimulating transcription as well as translation of cancer related genes. Our previous study demonstrated that α6β4 stimulates translation initiation of survival factors such as VEGF by activating mTOR pathway. However, the immediate early signaling events that link α6β4 to mTOR activation needs to be defined.ResultsIn the current studies, we demonstrated that c-Src is an immediate early signaling molecule that acts upstream of α6β4 dependent mTOR activation and subsequent translation of VEGF in MDA-MB-435/β4 and MDA-MB-231 cancer cells. m7GTP-Sepharose–binding assay revealed that Src activity is required to form eIF4F complex which is necessary for Cap-dependent translation in α6β4 expressing human cancer cells.ConclusionsOverall, our studies suggest that integrin β4 and c-Src activation is important early signaling events to lead mTOR activation and cap-dependent translation of VEGF.

Mnk mediates integrin (α6β4) dependent eIF4E phosphorylation and translation of VEGF mRNA

It was previously shown that integrin α6β4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6β4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6β4 and mediates the α6β4 signaling, important for translational control. Integrin α6β4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6β4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6β4 level is high. Mol Cancer Res; 8(12); 1571–8. ©2010 AACR.

Selective Inhibitors of Nuclear Export (SINE) compounds block proliferation and migration of triple negative breast cancer cells by restoring expression of ARRDC3

Arrestin-related domain-containing protein-3 (ARRDC3) is one of 6 mammalian arrestins, which suppresses metastasis by inducing degradation of phosphorylated β2-adrenergic receptor (β2 AR) and integrin β4 (ITG β4). Our previous studies demonstrated that expression of ARRDC3 is epigentically silenced in Triple Negative Breast Cancer (TNBC) cells, and the forced expression of ARRDC3 significantly reduced the invasive potential of TNBC cells. In the current study, we found that Selective Inhibitors of Nuclear Export (SINE) compounds (KPT-185 and selinexor (KPT-330)) restore ARRDC3 expression in TNBC cell lines (MDA-MB-231 and MDA-MB-468) at both the mRNA and protein level in a dose and time course dependent manner. SINE compounds inhibit the proliferation, pro-invasive migration and anchorage independent growth of the TNBC cells by restoring ARRDC3 expression. We found that ARRDC3 expression is lower in TNBC cell lines than those of luminal breast cancer cell lines, and inversely correlated with IC50s of selinexor. Analysis of tissue microarray confirmed that ARRDC3 expression in patient samples is significantly lower in the majority of TNBC tumors relative to normal tissue. In vivo, selinexor inhibited the tumor growth of MDA-MB-231 xenografts by nearly 100% compared with vehicle treated animals. Furthermore, immunohistochemical analysis of TNBC tumors from selinexor treated mice revealed increased ARRDC3 expression versus vehicle treated animals. Our results suggest that restoration of ARRDC3 expression is an important antineoplastic mechanism of SINE compounds in TNBC, and therefore selinexor could be an effective treatment option for breast tumors with down-regulated ARRDC3.Arrestin-related domain-containing protein-3 (ARRDC3) is one of 6 mammalian arrestins, which suppresses metastasis by inducing degradation of phosphorylated β2-adrenergic receptor (β2 AR) and integrin β4 (ITG β4). Our previous studies demonstrated that expression of ARRDC3 is epigentically silenced in Triple Negative Breast Cancer (TNBC) cells, and the forced expression of ARRDC3 significantly reduced the invasive potential of TNBC cells. In the current study, we found that Selective Inhibitors of Nuclear Export (SINE) compounds (KPT-185 and selinexor (KPT-330)) restore ARRDC3 expression in TNBC cell lines (MDA-MB-231 and MDA-MB-468) at both the mRNA and protein level in a dose and time course dependent manner. SINE compounds inhibit the proliferation, pro-invasive migration and anchorage independent growth of the TNBC cells by restoring ARRDC3 expression. We found that ARRDC3 expression is lower in TNBC cell lines than those of luminal breast cancer cell lines, and inversely correlated with IC50s of selinexor. Analysis of tissue microarray confirmed that ARRDC3 expression in patient samples is significantly lower in the majority of TNBC tumors relative to normal tissue. In vivo, selinexor inhibited the tumor growth of MDA-MB-231 xenografts by nearly 100% compared with vehicle treated animals. Furthermore, immunohistochemical analysis of TNBC tumors from selinexor treated mice revealed increased ARRDC3 expression versus vehicle treated animals. Our results suggest that restoration of ARRDC3 expression is an important antineoplastic mechanism of SINE compounds in TNBC, and therefore selinexor could be an effective treatment option for breast tumors with down-regulated ARRDC3.