Correction: Soung et al. Therapeutic Potential of Chemically Modified MiR-489 in Triple-Negative Breast Cancers. Cancers 2020, 12, 2209.

In the original publication [...].

Combination Treatment Strategies to Overcome PARP Inhibitor Resistance.

Poly(ADP-ribose) polymerase (PARP) enzymes have been shown to be essential for DNA repair pathways, including homologous recombination repair (HRR). Cancers with HRR defects (e.g., BRCA1 and BRCA2 mutations) are targets for PARP inhibitors (PARPis) based on the exploitation of "synthetic lethality". As a result, PARPis offer a promising treatment option for advanced ovarian and breast cancers with deficiencies in HRR. However, acquired resistance to PARPis has been reported for most tumors, and not all patients with BRCA1/2 mutations respond to PARPis. Therefore, the formulation of effective treatment strategies to overcome resistance to PARPis is urgently necessary. This review summarizes the molecular mechanism of therapeutic action and resistance to PARPis, in addition to emerging combination treatment options involving PARPis.

The Sensitization of Triple-Negative Breast Cancers to Poly ADP Ribose Polymerase Inhibition Independent of BRCA1/2 Mutation Status by Chemically Modified microRNA-489.

Chemoresistance and inefficient therapeutic efficacies in triple-negative breast cancers (TNBCs) are among the major clinical problems in breast cancers. A potential new method to sensitize these tumors to current treatment options is, therefore, urgent and necessary. Our previous studies demonstrated that miR-489 serves as one of the top tumor-suppressing miRs and features downregulated expression in metastatic TNBCs and that the restoration of miR-489 expression in TNBCs effectively inhibits the metastatic potentials of TNBCs both in vitro and in vivo. The chemical modification of miR-489 (CMM489) through the replacement of uracil with 5-FU further enhances the therapeutic potential of miR-489. In the present study, we tested the effects of CMM489 in synergizing DNA damage response (DDR) inhibitors such as PARP inhibitors. CMM489 is particularly effective in sensitizing TNBC cell lines with inherent resistance to PARP inhibitors regardless of BRCA mutation status. One of the anti-cancer mechanisms through which CMM489 synergizes with PARP inhibitors is the blockade of homologous recombination (HR) in TNBC cells upon DNA damage. The results of this study highlight the potential use of CMM489 in combination treatments with PARP inhibitors in TNBCs.

Therapeutic Potential of Chemically Modified miR-489 in Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) lack ER, PR and her2 receptors that are targets of common breast cancer therapies with poor prognosis due to their high rates of metastasis and chemoresistance. Based on our previous studies that epigenetic silencing of a potential metastasis suppressor, arrestin domain-containing 3 (ARRDC3), is linked to the aggressive nature of TNBCs, we identified a sub-group of tumor suppressing miRNAs whose expressions were significantly up-regulated by ARRDC3 over-expression in TNBC cells. Among these tumor suppressing miRs, we found that miR-489 is most anti-proliferative in TNBC cells. miR-489 also blocked DNA damaging responses (DDRs) in TNBC cells. To define the mechanism by which miR-489 inhibits TNBC cell functions, we screened the potential target genes of miR-489 and identified MDC-1 and SUZ-12 as novel target genes of miR-489 in TNBC cells. To further exploit the therapeutic potentials of miR-489 in TNBC models, we chemically modified the guide strand of miR-489 (CMM489) by replacing Uracil with 5-fluorouracil (5-FU) so that tumor suppressor (miR-489) and DNA damaging (5-FU) components are combined into a single agent as a novel drug candidate for TNBCs. Our studies demonstrated that CMM489 shows superior effects over miR-489 or 5-FU in inhibition of TNBC cell proliferation and tumor progression, suggesting its therapeutic efficacy in TNBC models.

Arrestin Domain Containing 3 Reverses Epithelial to Mesenchymal Transition and Chemo-Resistance of TNBC Cells by Up-Regulating Expression of miR-200b.

Our previous studies demonstrated the importance of arrestin domain containing 3 (ARRDC3), a metastasis suppressor, in inhibiting invasive and metastatic potential of triple negative breast cancer (TNBC) in vitro and in vivo. However, little is known about ARRDC3 mediated transcriptional control and its target genes that are implicated in its metastatic suppressing activity. In this study, we used miRNA array and subsequent functional analyses to identify miRNAs whose expression are significantly regulated by ARRDC3 in TNBC cells. We identified miR-200b as a major target gene of ARRDC3. miR-200b played an essential role in mediating ARRDC3 dependent reversal of EMT phenotypes and chemo-resistance to DNA damaging agents in TNBC cells. Expression of miR-200b also increased the expression of ARRDC3 as well in TNBC cells, suggesting a positive feedback loop between these two molecules. In addition, we combined the therapeutic powers of miR-200b and 5-fluorourancil (5-FU) into a single compound (5-FU-miR-200b) to maximize the synergistic effects of these compounds. Chemically modified miR-200b (5-FU-miR-200b mimic) was more effective in inhibiting metastatic potentials of TNBC cells than unmodified miR-200b and does not require transfection reagents, implying its therapeutic potential in TNBC. Our studies showed the importance of therapeutic targeting ARRDC3/miR-200b pathway in TNBC.

The Role of Arrestin Domain-Containing 3 in Regulating Endocytic Recycling and Extracellular Vesicle Sorting of Integrin ß4 in Breast Cancer.

Despite the established role of integrin ß4 (ITG ß4) in breast cancer progression, the importance of endocytic recycling of ITG ß4 and its regulatory mechanism are poorly understood. Here, we found that a sub-population of ITG ß4 is sorted into early endosomes, recycled back to the plasma membrane, and secreted in the form of extracellular vesicles (EVs) upon EGF treatment in triple negative breast cancer (TNBC) cells. A metastasis suppressor, ARRDC3 (arrestin domain-containing 3) prevents EGF-driven endocytic recycling of ITG ß4 by inducing NEDD4-dependent ubiquitination of ITG ß4 and targeting endosomal ITG ß4 into lysosomes. Endocytic recycling of ITG ß4 is linked to sorting of ITG ß4 into EVs (ITG ß4+ EVs). ITG ß4+ EVs are mainly detectable from supernatants of TNBC cells and their production is inhibited by ARRDC3 expression. ARRDC3 reduces the metastatic potentials of breast cancer cell-derived EVs by reducing ITG ß4 levels in EVs. Overall, current studies provide novel mechanistic insights on the regulatory mechanism of ITG ß4 recycling, and its importance in invasive potentials of TNBC EVs, thus providing the basis for therapeutic targeting of the ARRDC3/ITG ß4 pathway in TNBC.

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 ß2-adrenergic receptor (ß2 AR) and integrin ß4 (ITG ß4). 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.

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.

Emerging roles of exosomes in cancer invasion and metastasis.

Recent evidence has indicated that nano-sized vesicles called "exosomes" mediate the interaction between cancer cells and their microenvironment and play a critical role in the development of cancers. Exosomes contain cargo consisting of proteins, lipids, mRNAs, and microRNAs that can be delivered to different types of cells in nascent as well as distant locations. Cancer cell-derived exosomes (CCEs) have been identified in body fluids such as urine, plasma, and saliva from patients with cancer. Although their content depends on tumor type and stage, CCEs merit consideration as prognostic and diagnostic markers, as vehicles for drug delivery, and as potential therapeutic targets because they could transport various oncogenic elements. In this review, we summarize recent advances regarding the role of CCEs in cancer invasion and metastasis, as well as its potential clinical applications.

Curcumin Prevents Palmitoylation of Integrin ß4 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 ß4 (ITG ß4)-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 ß4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG ß4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG ß4 palmitoylation in invasive breast cancer cells. Through studies of ITG ß4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG ß4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG ß4 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 ß4 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 (α6ß4) dependent translational control.

BACKGROUND: Integrin α6ß4 contributes to cancer progression by stimulating transcription as well as translation of cancer related genes. Our previous study demonstrated that α6ß4 stimulates translation initiation of survival factors such as VEGF by activating mTOR pathway. However, the immediate early signaling events that link α6ß4 to mTOR activation needs to be defined. RESULTS: In the current studies, we demonstrated that c-Src is an immediate early signaling molecule that acts upstream of α6ß4 dependent mTOR activation and subsequent translation of VEGF in MDA-MB-435/ß4 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 α6ß4 expressing human cancer cells. CONCLUSIONS: Overall, our studies suggest that integrin ß4 and c-Src activation is important early signaling events to lead mTOR activation and cap-dependent translation of VEGF.

Somatic mutations of caspase-2 gene in gastric and colorectal cancers.

There is mounting evidence that evasion of apoptosis is a hallmark of cancer. Caspase-2, which plays roles in both extrinsic and intrinsic apoptosis pathways, is considered a candidate tumor suppressor. The aim of this study was to explore the possibility that genetic alterations of caspase-2 gene are present in human cancers. In this study, we analyzed the entire coding sequences of human caspase-2 gene for the detection of somatic point mutations in 90 gastric carcinomas and 100 colorectal carcinomas by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Of the cancers analyzed, two gastric cancers (2/90; 2.2%) and two colorectal cancers (2/100; 2.0%) harbored somatic missense mutations of caspase-2. The mutations consisted of p.V46M (at prodomain), p.S157L (at prodomain), p.R357K (at p13 subunit), and p.R397L (at p13 subunit). We expressed these tumor-derived mutants in 293 T cells and found that three of the mutants decreased cell death activity of caspase-2. Our data indicate that somatic mutation of caspase-2 is rare in gastric and colorectal carcinomas. However, functional data of the caspase-2 mutations also suggest that caspase-2 gene mutation might affect the pathogenesis of some gastric and colorectal cancers by inactivating cell death function of caspase-2.

Curcumin inhibition of the functional interaction between integrin α6ß4 and the epidermal growth factor receptor.

The functional interaction between integrin α6ß4 and growth factor receptors has been implicated in key signaling pathways important for cancer cell function. However, few attempts have been made to selectively target this interaction for therapeutic intervention. Previous studies showed that curcumin, a yellow pigment isolated from turmeric, inhibits integrin α6ß4 signaling important for breast carcinoma cell motility and invasion, but the mechanism is not currently known. To address this issue, we tested the hypothesis that curcumin inhibits the functional interaction between α6ß4 and the epidermal growth factor receptor (EGFR). In this study, we found that curcumin disrupts functional and physical interactions between α6ß4 and EGFR, and blocks α6ß4/EGFR-dependent functions of carcinoma cells expressing the signaling competent form of α6ß4. We further showed that curcumin inhibits EGF-dependent mobilization of α6ß4 from hemidesmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby prevents α6ß4 distribution to lipid rafts where functional interactions between α6ß4 and EGFR occur. These data suggest a novel paradigm in which curcumin inhibits α6ß4 signaling and functions by altering intracellular localization of α6ß4, thus preventing its association with signaling receptors such as EGFR.

Role of α6ß4 integrin in cell motility, invasion and metastasis of mammary tumors.

Integrin α6ß4 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 α6ß4 in their progression. The tumor microenvironment is also known to enhance the signaling competence of α6ß4 through functional and physical interactions with other receptors. In this review, we discuss the biological mechanisms by which integrin α6ß4 promotes carcinoma cell motility and invasion that leads to mammary tumor progression.

Effect of {beta}4 integrin knockdown by RNA interference in anaplastic thyroid carcinoma.

BACKGROUND: Integrin α6ß4 is a known tumor antigen; however, its function in different subtypes of thyroid cancer is not known. This study reports that α6ß4 expression is selectively up-regulated in anaplastic thyroid cancer (ATC) cells, the most malignant subtype of human thyroid cancer. MATERIALS AND METHODS: To assess the contribution of α6ß4 in ATC progression, cell proliferation, motility and soft agar assay were performed in vitro and a xenograft tumor growth assay was performed in vivo. RESULTS: Knockdown of ß4 integrin subunit expression by shRNA in ATC cells reduced the proliferation, migration, and anchorage-independent growth of ATC cells in vitro and xenograft tumor growth in vivo. CONCLUSION: These data suggest that integrin α6ß4 contributes to the development of aggressive forms of thyroid cancer with poor prognostic potential, such as ATC, and thus may be a novel therapeutic target for the treatment for this subtype of thyroid cancer.

Mnk mediates integrin α6ß4-dependent eIF4E phosphorylation and translation of VEGF mRNA.

It was previously shown that integrin α6ß4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6ß4 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 α6ß4 and mediates the α6ß4 signaling, important for translational control. Integrin α6ß4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6ß4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6ß4 level is high.

Crosstalk between integrin and receptor tyrosine kinase signaling in breast carcinoma progression.

This review explored the mechanism of breast carcinoma progression by focusing on integrins and receptor tyrosine kinases (or growth factor receptors). While the primary role of integrins was previously thought to be solely as mediators of adhesive interactions between cells and extracellular matrices, it is now believed that integrins also regulate signaling pathways that control cancer cell growth, survival, and invasion. A large body of evidence suggests that the cooperation between integrin and receptor tyrosine kinase signaling regulates certain signaling functions that are important for cancer progression. Recent developments on the crosstalk between integrins and receptor tyrosine kinases, and its implication in mammary tumor progression, are discussed.

Mutant p53 (G199V) gains antiapoptotic function through signal transducer and activator of transcription 3 in anaplastic thyroid cancer cells.

In the present study, we identified a missense mutation (G199V) in KAT-18 cell line established from primary cultures of anaplastic thyroid cancer (ATC). Notably, knockdown of this mutant (mt) p53 reduced cell viability and exerted antitumor activity equivalent to high doses of several chemotherapeutic agents. We showed that p53 knockdown had an antitumor effect via the induction of apoptosis. We further examined the underlying mechanism by which mt p53 (G199V) gains antiapoptotic function in KAT-18 cells. Microarray analysis revealed that p53 knockdown modified the expression of numerous apoptosis-related genes. Importantly, p53 knockdown led to downregulation of signal transducer and activator of transcription-3 (STAT3) gene expression. We further observed that p53 knockdown induced the downregulation of STAT3 protein. We also observed that a STAT3 inhibitor augmented the reduction of cell viability induced by p53 knockdown, whereas interleukin-6 treatment alleviated this effect. In addition, overexpression of STAT3 protected ATC cells against cell death induced by p53 knockdown. Taken together, these data show that mt p53 (G199V) gains antiapoptotic function mediated by STAT3 in ATC cells. Inhibition of the function of mt p53 (G199V) could be a novel and useful therapeutic strategy for decreasing the extent and severity of toxicity due to chemotherapeutic agents.

Integrin (alpha6beta4) signals through Src to increase expression of S100A4, a metastasis-promoting factor: implications for cancer cell invasion.

Integrin alpha6beta4 is linked to cancer cell motility and invasion in aggressive and metastatic cancer cells. In this study, we showed that expression of the beta4 integrin in MDA-MB-435 cancer cells (MDA-MB-435/beta4) 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 beta4 integrin expression in MDA-MB-231 breast carcinoma cells by shRNA reduced the level of S100A4 expression. The mechanism by which alpha6beta4 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 beta4 integrin subunit, is essential for alpha6beta4-dependent S100A4 expression. Reduction of S100A4 expression by shRNA blocked migration, invasion, and anchorage-independent growth of MDA-MB-435/beta4, SUM-159, and MDA-MB-231 cells. These studies define a novel mechanism by which integrin alpha6beta4 promotes cancer cell motility and invasion, and provides insight into how S100A4 expression is regulated in cancer cells.