K-RAS Acts as a Critical Regulator of CD44 to Promote the Invasiveness and Stemness of GBM in Response to Ionizing Radiation.

Radiation therapy is a current standard-of-care treatment and is used widely for GBM patients. However, radiation therapy still remains a significant barrier to getting a successful outcome due to the therapeutic resistance and tumor recurrence. Understanding the underlying mechanisms of this resistance and recurrence would provide an efficient approach for improving the therapy for GBM treatment. Here, we identified a regulatory mechanism of CD44 which induces infiltration and mesenchymal shift of GBM. Ionizing radiation (IR)-induced K-RAS/ERK signaling activation elevates CD44 expression through downregulation of miR-202 and miR-185 expression. High expression of CD44 promotes SRC activation to induce cancer stemness and EMT features of GBM cells. In this study, we demonstrate that the K-RAS/ERK/CD44 axis is a key mechanism in regulating mesenchymal shift of GBM cells after irradiation. These findings suggest that blocking the K-RAS activation or CD44 expression could provide an efficient way for GBM treatment.

Persistent activation of STAT3 by PIM2-driven positive feedback loop for epithelial-mesenchymal transition in breast cancer.

Metastasis of breast cancer is promoted by epithelial-mesenchymal transition (EMT). Emerging evidence suggests that STAT3 is a critical signaling node in EMT and is constitutively activated in many carcinomas, including breast cancer. However, its signaling mechanisms underlying persistent activation of STAT3 associated with EMT remain obscure. Here, we report that PIM2 promotes activation of STAT3 through induction of cytokines. Activation of STAT3 caused an increase in PIM2 expression, implicating a positive feedback loop between PIM2 and STAT3. In agreement, targeting of either PIM2, STAT3 or PIM2-dependent cytokines suppressed EMT-associated migratory and invasive properties through inhibition of ZEB1. Taken together, our findings identify the signaling mechanisms underlying the persistent activation of STAT3 and the oncogenic role of PIM2 in EMT in breast cancer.

Radiation promotes malignant phenotypes through SRC in breast cancer cells.

Despite the fact that ionizing radiation (IR) is widely used as a standard treatment for breast cancer, much evidence suggests that IR paradoxically promotes cancer malignancy. However, the molecular mechanisms underlying radiation-induced cancer progression remain obscure. Here, we report that irradiation activates SRC signaling among SRC family kinase proteins, thereby promoting malignant phenotypes such as invasiveness, expansion of the cancer stem-like cell population, and resistance to anticancer agents in breast cancer cells. Importantly, radiation-activated SRC induced SLUG expression and caused epithelial-mesenchymal cell transition through phosphatidylinositol 3-kinase/protein kinase B and p38 MAPK signaling. In agreement, either inhibition of SRC or downstream signaling of p38 MAPK or protein kinase B effectively attenuated radiation-induced epithelial-mesenchymal cell transition along with an increase in the cancer stem-like cell population. In addition, downregulation of SRC also abolished radiation-acquired resistance of breast cancer cells to anticancer agents such as cisplatin, etoposide, paclitaxel, and IR. Taken together, our findings suggest that combining radiotherapy with targeting of SRC might attenuate the harmful effects of radiation and enhance the efficacy of breast cancer treatment.

Phloroglucinol suppresses metastatic ability of breast cancer cells by inhibition of epithelial-mesenchymal cell transition.

Metastasis is a challenging clinical problem and the primary cause of death in breast cancer patients. However, there is no therapeutic agent against metastasis of breast cancer cells. Here we report that phloroglucinol, a natural phlorotannin component of brown algae suppresses metastatic ability of breast cancer cells. Treatment with phloroglucinol effectively inhibited mesenchymal phenotypes of basal type breast cancer cells through downregulation of SLUG without causing a cytotoxic effect. Importantly, phloroglucinol decreased SLUG through inhibition of PI3K/AKT and RAS/RAF-1/ERK signaling. In agreement with in vitro data, phloroglucinol was also effective against in vivo metastasis of breast cancer cells, drastically suppressing their metastatic ability to lungs, and extending the survival time of mice. Collectively, our findings demonstrate a novel anticancer activity of phloroglucinol against metastasis of breast cancer cells, implicating its clinical relevance.

PTTG1 oncogene promotes tumor malignancy via epithelial to mesenchymal transition and expansion of cancer stem cell population.

The prognosis of breast cancer patients is related to the degree of metastasis. However, the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize at a distant site are not entirely understood. Here, we analyzed expression levels of pituitary tumor-transforming gene-1 (PTTG1), a relatively uncharacterized oncoprotein, in patient-derived breast cancer tissues with corresponding normal breast tissues. We found that PTTG1 is highly expressed in breast cancer patients, compared with normal tissues. Also, PTTG1 expression levels were correlated with the degree of malignancy in breast cancer cell lines; the more migratory and invasive cancer cell lines MDA-MB-231 and BT549 displayed the higher expression levels of PTTG1 than the less migratory and invasive MCF7 and SK-BR3 and normal MCF10A cell lines. By modulating PTTG1 expression levels, we found that PTTG1 enhances the migratory and invasive properties of breast cancer cells by inducing epithelial to mesenchymal transition, as evidenced by altered morphology and epithelial/mesenchymal cell marker expression patterns and up-regulation of the transcription factor Snail. Notably, down-regulation of PTTG1 also suppressed cancer stem cell population in BT549 cells by decreasing self-renewing ability and tumorigenic capacity, accompanying decreasing CD44(high) CD24(low) cells and Sox2 expression. Up-regulation of PTTG1 had the opposite effects, increasing sphere-forming ability and Sox2 expression. Importantly, PTTG1-mediated malignant tumor properties were due, at least in part, to activation of AKT, known to be a key regulator of both EMT and stemness in cancer cells. Collectively, these results suggest that PTTG1 may represent a new therapeutic target for malignant breast cancer.

Fractionated radiation-induced nitric oxide promotes expansion of glioma stem-like cells.

Glioblastoma remains an incurable brain disease due to the prevalence of its recurrence. Considerable evidence suggests that glioma stem-like cells are responsible for glioma relapse after treatment, which commonly involves ionizing radiation. Here, we found that fractionated ionizing radiation (2 Gy/day for 3 days) induced glioma stem-like cell expansion and resistance to anticancer treatment such as cisplatin (50 µM) or taxol (500 nM), or by ionizing radiation (10 Gy) in both glioma cell lines (U87, U373) and patient-derived glioma cells. Of note, concomitant increase of nitric oxide production occurred with the radiation-induced increase of the glioma stem-like cell population through upregulation of inducible nitric oxide synthase (iNOS). In line with this observation, downregulation of iNOS effectively reduced the glioma stem-like cell population and decreased resistance to anticancer treatment. Collectively, our results suggest that targeting iNOS in combination with ionizing radiation might increase the efficacy of radiotherapy for glioma treatment.

A new 2-pyrone derivative, 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one, suppresses stemness in glioma stem-like cells.

Glioma cells with stem cell properties, termed glioma stem-like cells (GSCs), have been linked to tumor formation, maintenance, and progression and are responsible for the failure of chemotherapy and radiotherapy. Because conventional glioma treatments often fail to eliminate GSCs completely, residual surviving GSCs are able to repopulate the tumor. Compounds that target GSCs might be helpful in overcoming resistance to anticancer treatments in human brain tumors. In this study, we showed that 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one (BHP), a new 2-pyrone derivative, suppressed the maintenance of the GSC population in both a glioma cell line and patient-derived glioma cells. Treatment of GSCs with BHP effectively inhibited sphere formation and suppressed the CD133(+) cell population. Treatment with BHP also suppressed expression of the stemness-regulating transcription factors Sox2, Notch2, and ß-catenin in sphere-cultured glioma cells. Treatment of GSCs with BHP significantly suppressed two fundamental characteristics of cancer stem cells: self-renewal and tumorigenicity. BHP treatment dramatically inhibited clone-forming ability at the single-cell level and suppressed in vivo tumor formation. BHP markedly inhibited both phosphoinositide 3-kinase/Akt and Ras/Raf-1/extracellular signal-regulated kinase signaling, which suggests that one or both of these pathways are involved in BHP-induced suppression of GSCs. In addition, treatment with BHP effectively sensitized GSCs to chemotherapy and radiotherapy. Taken together, these results indicate that BHP targets GSCs and enhances their sensitivity to anticancer treatments and suggest that BHP treatment may be useful for treating brain tumors by eliminating GSCs.

Glioblastoma-educated mesenchymal stem-like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment.

BACKGROUND: The biological function of mesenchymal stem-like cells (MSLCs), a type of stromal cells, in the regulation of the tumour microenvironment is unclear. Here, we investigated the molecular mechanisms underlying extracellular matrix (ECM) remodelling and crosstalk between MSLCs and glioblastomas (GBMs) in tumour progression. METHODS: In vitro and in vivo co-culture systems were used to analyze ECM remodelling and GBM infiltration. In addition, clinical databases, samples from patients with GBM and a xenografted mouse model of GBM were used. RESULTS: Previous studies have shown that the survival of patients with GBM from whom MSLCs could be isolated is substantially shorter than that of patients from whom MSLCs could not be isolated. Therefore, we determined the correlation between changes in ECM-related gene expression in MSLC-isolatable patients with that in MSLC non-isolatable patients using gene set enrichment analysis (GSEA). We found that lysyl oxidase (LOX) and COL1A1 expressions increased in MSLCs via GBM-derived clusters of differentiation 40 ligand (CD40L). Mechanistically, MSLCs are reprogrammed by the CD40L/CD40/NFκB2 signalling axis to build a tumour infiltrative microenvironment involving collagen crosslinking. Importantly, blocking of CD40L by a neutralizing antibody-suppressed LOX expression and ECM remodelling, decreasing GBM infiltration in mouse xenograft models. Clinically, high expression of CD40L, clusters of differentiation 40 (CD40) and LOX correlated with poor survival in patients with glioma. This indicated that GBM-educated MSLCs promote GBM infiltration via ECM remodelling in the tumour microenvironment. CONCLUSION: Our findings provide mechanistic insights into the pro-infiltrative tumour microenvironment produced by GBM-educated MSLCs and highlight a potential therapeutic target that can be used for suppressing GBM infiltration.

Soluble ICAM-1 a Pivotal Communicator between Tumors and Macrophages, Promotes Mesenchymal Shift of Glioblastoma.

Despite aggressive clinical treatment, recurrence of glioblastoma multiforme (GBM) is unavoidable, and the clinical outcome is still poor. A convincing explanation is the phenotypic transition of GBM cells upon aggressive treatment such as radiotherapy. However, the microenvironmental factors contributing to GBM recurrence after treatment remain unexplored. Here, it is shown that radiation-treated GBM cells produce soluble intercellular adhesion molecule-1 (sICAM-1) which stimulates the infiltration of macrophages, consequently enriching the tumor microenvironment with inflammatory macrophages. Acting as a paracrine factor, tumor-derived sICAM-1 induces macrophages to secrete wingless-type MMTV integration site family, member 3A (WNT3A), which promotes a mesenchymal shift of GBM cells. In addition, blockade of either sICAM-1 or WNT3A diminishes the harmful effect of radiation on tumor progression. Collectively, the findings indicate that cellular crosstalk between GBM and macrophage through sICAM-1-WNT3A oncogenic route is involved in the mesenchymal shift of GBM cells after radiation, and suggest that radiotherapy combined with sICAM-1 targeted inhibition would improve the clinical outcome of GBM patients.

Continuous Separation of Circulating Tumor Cells from Whole Blood Using a Slanted Weir Microfluidic Device.

The separation of circulating tumor cells (CTCs) from the peripheral blood is an important issue that has been highlighted because of their high clinical potential. However, techniques that depend solely on tumor-specific surface molecules or just the larger size of CTCs are limited by tumor heterogeneity. Here, we present a slanted weir microfluidic device that utilizes the size and deformability of CTCs to separate them from the unprocessed whole blood. By testing its ability using a highly invasive breast cancer cell line, our device achieved a 97% separation efficiency, while showing an 8-log depletion of erythrocytes and 5.6-log depletion of leukocytes. We also developed an image analysis tool that was able to characterize the various morphologies and differing deformability of the separating cells. From the results, we believe our system possesses a high potential for liquid biopsy, aiding future cancer research.

Deterministic Capture of Individual Circulating Tumor Cells Using a Flow-Restricted Microfluidic Trap Array.

Circulating tumor cells (CTCs) are regarded as a strong biomarker which includes clinically valuable information. However, CTCs are very rare and require precise separation and detection for effective clinical applications. Furthermore, downstream analysis has become necessary to identify the distinct sub-population of CTCs that causes metastasis. Here, we report a flow-restricted microfluidic trap array capable of deterministic single-cell capture of CTCs. The extent of flow restriction, correlating with the device geometry, was then optimized using a highly invasive breast cancer cell line (LM2 MDA-MB-231) to achieve 97% capture efficiency with a single-cell capture rate of 99%. Single-cell capture of CTCs from mice with full-blown metastasis was also demonstrated. The single-CTC capturing ability of the flow-restricted trap array not only showed cell enumerating ability but also high prospects for application in future automated downstream analysis.

MerTK mediates STAT3-KRAS/SRC-signaling axis for glioma stem cell maintenance.

Receptor tyrosine kinase Mer (MerTK) has been shown to be highly expressed in Glioblastoma multiforme (GBM) in comparison to its healthy counterpart and is implicated in brain tumorigenesis. Clarifying the underlying mechanism of MerTK induced invasiveness would result in novel strategies to improve patient's response to chemotherapeutics. In vitro and in vivo assays were performed to examine the functional role of cancer stem sell (CSC) maintenance in MerTK associated invasiveness. In this article, we demonstrate that apart from GBM cells, MerTK is also upregulated in GBM stem-like cells and associated with an increased infiltrative potential of brain tumors in vivo. Silencing of MerTK suppressed the self-renewal of patient-derived GBM stem-like cells. The signaling mechanisms by which MerTK contributes to CSC maintenance have largely been obscure. Molecular analyses revealed that high expression of the signal transducer and activator of transcription 3 (STAT3)- Kirsten rat sarcoma viral oncogene homolog (KRAS) and proto-oncogene tyrosine-protein kinase SRC axis supports MerTK-induced CSC maintenance in GBM spheroids. Furthermore, a short-hairpin RNA-mediated MerTK knockdown effectively blocked invasiveness and N-cadherin expression in mouse xenografts. Collectively, our results uncover a critical function of MerTK in CSC maintenance. Considering the low basal level of MerTK expression in healthy brain cells, evaluation of MerTK as a therapeutic target should advance the research into better therapeutics for GBM.

FYN promotes mesenchymal phenotypes of basal type breast cancer cells through STAT5/NOTCH2 signaling node.

Basal type breast cancer is the most aggressive and has mesenchymal features with a high metastatic ability. However, the signaling node that determines the basal type features in breast cancer remains obscure. Here, we report that FYN among SRC family kinases is required for the maintenance of basal type breast cancer subtype. Importantly, FYN enhanced NOTCH2 activation in basal type breast cancer cells through STAT5-mediated upregulation of Jagged-1 and DLL4 NOTCH ligands, thereby contributed to mesenchymal phenotypes. In addition, we found that high levels of FYN persist in basal type breast cancer cells by a positive feedback loop between FYN and STAT5. FYN interacted directly with STAT5 and increased p-STAT5 that further acts as a transcription factor for FYN. Taken together, our findings demonstrate a pivotal role of FYN and its downstream effectors in maintaining the basal type features in breast cancer.

Proinvasive extracellular matrix remodeling in tumor microenvironment in response to radiation.

Ionizing radiation is widely used for patient with glioblastoma (GBM). However, the effect of radiation on patient survival is marginal and upon recurrence tumors frequently shift toward mesenchymal subtype adopting invasiveness. Here, we show that ionizing radiation affects biomechanical tension in GBM microenvironment and provides proinvasive extracellular signaling cue, hyaluronic acid (HA)-rich condition. In response to radiation, HA production was increased in GBM cells by HA synthase-2 (HAS2) that was transcriptionally upregulated by NF-ĸB. Notably, NF-ĸB was persistently activated by IL-1α-feedback loop, making HA abundance in tumor microenvironment after radiation. Radiation-induced HA abundance causally has been linked to invasiveness of GBM cells by generating movement track as an extracellular matrix, and by acting as a signaling ligand for CD44 receptor, leading to SRC activation, which is sufficient for mesenchymal shift of GBM cells. Collectively, our findings provide an explanation for the frequent brain tumor relapse after radiotherapy, and potential therapeutic targets to block mesenchymal shift upon relapse.

FBXL14 abolishes breast cancer progression by targeting CDCP1 for proteasomal degradation.

Understanding the molecular mechanisms that underlie the aggressive behavior and relapse of breast cancer may help in the development of novel therapeutic interventions. CUB-domain-containing protein 1 (CDCP1), a transmembrane adaptor protein, is highly maintained and required in the context of cellular metastatic potential in triple-negative breast cancer (TNBC). For this reason, gene expression levels of CDCP1 have been considered as a prognostic marker in TNBC. However, not rarely, transcript levels of genes do not reflect always the levels of proteins, due to the post-transcriptional regulation. Here we show that miR-17/20a control the FBXL14 E3 ligase, establishing FBXL14 as an upstream regulator of the CDCP1 pathway. FBXL14 acts as an novel interaction partner of CDCP1, and facilitates its ubiquitination and proteasomal degradation with an enhanced capacity to suppress CDCP1 protein stability that eventually prevents CDCP1 target genes involved in breast cancer metastasis. Our findings first time uncovers the regulatory mechanism of CDCP-1 protein stabilization, more predictable criteria than gene expression levels for prognosis of breast cancer patients.

Hollow hyaluronic acid particles by competition between adhesive and cohesive properties of catechol for anticancer drug carrier.

The marine mussel-inspired properties of catechol, adhesiveness and cohesiveness, have been applied with pH control to fabricate hollow particles using a silica core and catechol-modified hyaluronic acid (HA-CA) shell for an anticancer drug carrier. The competition between adhesive and cohesive properties of catechol with different pH values leads to various structures, a rough catechol modified HA (HA-CA) shell at pH 5.5, monodisperse spherical silica@HA-CA particles at pH 7.4, and an amorphous HA-CA layer at pH 8.5. The redox transition of catechol with pH is a key factor modulating the behavior of the HA-CA shell on the silica core, which induces strong adhesion of HA-CA to silica at pH 5.5 and structural hardness with cohesive coupling at pH 7.4. In addition, after core removal, the hollow HA-CA particles are followed by loading of anticancer drug, doxorubicin (DOX). DOX loaded HA-CA particles show pH-triggered release behavior and dramatic cytotoxic effect indicating that they are a promising novel anticancer drug carrier.

Regulation of FBXO4-mediated ICAM-1 protein stability in metastatic breast cancer.

Advanced or progressive cancers share common traits such as altered transcriptional modulation, genetic modification, and abnormal post-translational regulation. These processes influence protein stability and cellular activity. Intercellular adhesion molecule-1 (ICAM-1) is involved in the malignant progression of various human cancers, including breast, liver, renal, and pancreatic cancers, but protein stability has not been deal with in metastatic breast cancer. Additionally, the relevance of the stability maintenance of ICAM-1 protein remains obscure. Here, we identified a novel interaction of E3 ligase FBXO4 that is specifically presented to ICAM-1. To understand how FBXO4 modulates ICAM-1 stability, we investigated ICAM-1-overexpressing or knockdown metastatic/non-metastatic breast cancers. ICAM-1 was found to influence tumor progression and metastasis, whereas FBXO4 regulated aggressive tumorigenic conditions. These results demonstrate that FBXO4 is a major regulator of ICAM-1 stability and that alterations in the stability of ICAM-1 can influence therapeutic outcome in metastatic cancer.

Tumor-associated mesenchymal stem-like cells provide extracellular signaling cue for invasiveness of glioblastoma cells.

Hyaluronic acid (HA) is abundant in tumor microenvironment and closely associated with invasiveness of glioblastoma (GBM) cells. However, the cellular mechanism underlying HA-rich microenvironment in GBM remains unexplored. Here, we show that tumor-associated mesenchymal stem-like cells (tMSLCs) contribute to abundance of hyaluronic acid (HA) in tumor microenvironment through HA synthase-2 (HAS2) induction, and thereby enhances invasiveness of GBM cells. In an autocrine manner, C5a secreted by tMSLCs activated ERK MAPK for HAS2 induction in tMSLCs. Importantly, HA acted as a signaling ligand of its cognate receptor RHAMM for intracellular signaling activation underlying invasiveness of GBM cells. Taken together, our study suggests that tMSLCs contribute to HA-rich proinvasive ECM microenvironment in GBM.

Radiation driven epithelial-mesenchymal transition is mediated by Notch signaling in breast cancer.

Epithelial to mesenchymal transition (EMT) is developmental process associated with cancer metastasis. Here, we found that breast carcinoma cells adopt epithelial-to-mesenchymal transition (EMT) in response to fractionated-radiation. Importantly, we show that Notch signaling is highly activated in fractionally-irradiated tumors as compared to non-irradiated tumors that are accompanied by an EMT. Moreover, we uncovered the mechanism of Notch-driven EMT, in which Notch enhanced EMT through IL-6/JAK/STAT3 signaling axis in mammary tumor cells. Collectively, we present converging evidence from our studies that Notch2 is a critical mediator of radiation-induced EMT and responsible for induced malignant tumor growth.

Low doses of PEG-coated gold nanoparticles sensitize solid tumors to cold plasma by blocking the PI3K/AKT-driven signaling axis to suppress cellular transformation by inhibiting growth and EMT.

Metastasis, the primary cause of tumor cell transformation, is often activated during cancer invasion and progression and is associated with poor therapeutic outcomes. The effects of combined treatments that included PEG-coated gold nanoparticles (GNP) and cold plasma on epithelial-mesenchymal transition (EMT) and the maintenance of cancer stem cells (CSC) have not been described so far. Here, we report that co-treatment with GNP and cold plasma inhibited proliferation in cancer cells by abolishing the activation of the PI3K/AKT signaling axis. In addition, co-treatment reversed EMT in solid tumor cells by reducing the secretion of a number of proteins, resulting in the upregulation of epithelial markers such as E-cadherin along with down-regulation of N-Cadherin, Slug and Zeb-1. The inhibition of the PI3K/AKT pathway and the reversal of EMT by co-treatment prevented tumor cells growth in solid tumors. Furthermore, we show that GNP and plasma also suppresses tumor growth by decreasing mesenchymal markers in tumor xenograft mice models. Importantly, co-treatment resulted in a substantial decrease in sphere formation and the self-renewal capacity of glioma-like stem cells. Together, these results indicate a direct link between a decrease of EMT and an increase in cell death in solid tumors following co-treatment with cold plasma and GNP.