Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas.

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.

EGFR is a master switch between immunosuppressive and immunoactive tumor microenvironment in inflammatory breast cancer.

Inflammatory breast cancer (IBC), the most aggressive breast cancer subtype, is driven by an immunosuppressive tumor microenvironment (TME). Current treatments for IBC have limited efficacy. In a clinical trial (NCT01036087), an anti-EGFR antibody combined with neoadjuvant chemotherapy produced the highest pathological complete response rate ever reported in patients with IBC having triple-negative receptor status. We determined the molecular and immunological mechanisms behind this superior clinical outcome. Using novel humanized IBC mouse models, we discovered that EGFR-targeted therapy remodels the IBC TME by increasing cytotoxic T cells and reducing immunosuppressive regulatory T cells and M2 macrophages. These changes were due to diminishing immunosuppressive chemokine expression regulated by transcription factor EGR1. We also showed that induction of an immunoactive IBC TME by an anti-EGFR antibody improved the antitumor efficacy of an anti-PD-L1 antibody. Our findings lay the foundation for clinical trials evaluating EGFR-targeted therapy combined with immune checkpoint inhibitors in patients with cancer.

Identification of the JNK-Active Triple-Negative Breast Cancer Cluster Associated With an Immunosuppressive Tumor Microenvironment.

BACKGROUND: Although an immunosuppressive tumor microenvironment (TME) is key for tumor progression, the molecular characteristics associated with the immunosuppressive TME remain unknown in triple-negative breast cancer (TNBC). Our previous functional proteomic study of TNBC tumors identified that C-JUN N-terminal kinase (JNK) pathway-related molecules were enriched in a cluster associated with the inflammatory pathway. However, the role of the JNK pathway in the TNBC TME is still unclear. METHODS: Transcriptomic analysis was conducted using The Cancer Genome Atlas datasets. The effect of JNK-IN-8, a covalent pan-JNK inhibitor, on TNBC tumor growth, lung metastasis, and the TME was measured in TNBC syngeneic mouse models (n = 13 per group). Tumor (n = 43) or serum (n = 46) samples from TNBC patients were analyzed using multiplex immunohistochemistry or Luminex assay. All statistical tests were 2-sided. RESULTS: CIBERSORT analysis revealed that TNBC patients with high phosphorylated JNK level (n = 47) had more regulatory T cell (Treg) infiltration than those with a low phosphorylated JNK level (n = 47) (P = .02). Inhibition of JNK signaling statistically significantly reduced tumor growth (P < .001) and tumor-infiltrating Tregs (P = .02) while increasing the infiltration of CD8+ T cells in TNBC mouse models through the reduction of C-C motif ligand 2 (CCL2). Tumor-associated macrophages were the predominant cells secreting CCL2, and inhibition of JNK signaling reduced CCL2 secretion of human primary macrophages. Moreover, in patients with TNBC (n = 43), those with high levels of CCL2+ tumor-associated macrophages had more Treg and less CD8+ T cell infiltration (P = .04), and the serum CCL2 level was associated with poor overall survival (hazard ratio = 2.65, 95% confidence interval = 1.29 to 5.44, P = .008) in TNBC patients (n = 46). CONCLUSIONS: The JNK/C-JUN/CCL2 axis contributes to TNBC aggressiveness via forming an immunosuppressive TME and can offer novel therapeutic strategies for TNBC.

Targeting Signaling Pathways in Inflammatory Breast Cancer.

Inflammatory breast cancer (IBC), although rare, is the most aggressive type of breast cancer. Only 2-4% of breast cancer cases are classified as IBC, but-owing to its high rate of metastasis and poor prognosis-8% to 10% of breast cancer-related mortality occur in patients with IBC. Currently, IBC-specific targeted therapies are not available, and there is a critical need for novel therapies derived via understanding novel targets. In this review, we summarize the biological functions of critical signaling pathways in the progression of IBC and the preclinical and clinical studies of targeting these pathways in IBC. We also discuss studies of crosstalk between several signaling pathways and the IBC tumor microenvironment.

20(R)-Ginsenoside Rg3 Influences Cancer Stem Cell Properties and the Epithelial-Mesenchymal Transition in Colorectal Cancer via the SNAIL Signaling Axis.

BACKGROUND: Cancer stem cells (CSCs) have been proposed as central drivers of cancer relapse in many cancers. In the present study, we investigated the inhibitory effect of 20(R)-Ginsenoside Rg3 (Rg3R), a major active component of ginseng saponin, on CSC-like cells and the Epithelial-Mesenchymal Transition (EMT) in colorectal cancer (CRC). METHODS: The effects of ginsenoside Rg3R on the colony-forming, migration, invasion, and wound-healing abilities of CRC cells were determined in HT29 and SW620 cell lines in vitro. Further, ginsenoside Rg3R was given intraperitoneally at 5mg/kg of mouse body weight to check its effect on the metastasis of CRC cells in vivo. RESULTS: Ginsenoside Rg3R significantly inhibited CSC properties, but did not affect cell proliferation. Moreover, ginsenoside Rg3R treatment significantly inhibited the motility of CRC cells based on migration, invasion, and wound-healing assays. The inhibitory effects of ginsenoside Rg3R on CRC are potentially mediated by significant down-regulation of the expression of stemness genes and EMT markers in CRC cells in a SNAIL-dependent manner. Furthermore, ginsenoside Rg3R treatment decreased both the number and size of tumor nodules in the liver, lung, and kidney tissues in a metastasis mouse model. CONCLUSION: These findings highlighted the potential use of ginsenoside Rg3R in clinical applications for colorectal cancer treatment.

In Vitro Production of Cartilage Tissue from Rabbit Bone Marrow-Derived Mesenchymal Stem Cells and Polycaprolactone Scaffold.

In vitro production of tissues or tissue engineering is a promising approach to produce artificial tissues for regenerative medicine. There are at least three important components of tissue engineering, including stem cells, scaffolds and growth factors. This study aimed to produce cartilage tissues in vitro from culture and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMMSCs), induced by chondrogenesis medium, on biodegradable polycaprolactone (PCL) scaffolds. BMMSCs were isolated from rabbit bone marrow according to the standard protocol. The adherence, proliferation and differentiation of BMMSCs on scaffolds were investigated using two scaffold systems: PCL scaffolds and collagen-coated PCL (PCL/col) scaffolds. The results showed that BMMSCs could attach and grow on both PCL and PCL/col scaffolds. However, the adhesion efficacy of BMMSCs on the PCL/col scaffolds was significantly better than on PCL scaffolds. Under induced conditions, BMMSCs on PLC/col scaffolds showed increased aggrecan accumulation and upregulated expression of chondrogenesis-associated genes (e.g. collagen type II, collagen type I, aggrecan and collagen type X) after 3, 7, 21 and 28 days of induction. These in vitro cartilage tissues could form mature chondrocyte-like cells after they were grafted into rabbits. The results suggest that use of BMMSCs in combination with polycaprolactone scaffolds and chondrogenesis medium can be a way to form in vitro cartilage tissue.

Interferon-induced transmembrane protein 1-mediated EGFR/SOX2 signaling axis is essential for progression of non-small cell lung cancer.

Emerging data indicate that interferon-induced transmembrane protein 1 (IFITM1) plays an important role in many cancers. However, it remains unclear whether IFITM1 is functionally indispensable in nonsmall cell lung cancer (NSCLC). Here, using NSCLC cell lines and patient-derived samples, we show that IFITM1 is essentially required for the progression of NSCLC in vitro and in vivo. Specifically, IFITM1 depletion resulted in a significant reduction in sphere formation, migration, and invasion of NSCLC cells in vitro; these events were inversely correlated with the ectopic expression of IFITM1. In addition, tumor development was significantly impaired in the absence of IFITM1 in vivo. Mechanistically, epidermal growth factor receptor/sex-determining region Y-box 2 (EGFR/SOX2) signaling axis was compromised in the absence of IFITM1, and the ectopic expression of SOX2 partially rescued the defects caused by IFITM1 depletion. More importantly, using 226 patient-derived samples, we demonstrate that a high level of IFITM1 expression is associated with a poor overall survival (OS) rate in adenocarcinoma but not in squamous cell carcinoma. Collectively, these data suggest that IFITM1 is a poor prognostic marker of adenocarcinoma and an attractive target to develop novel therapeutics for NSCLC.

Ginsenoside Rd Inhibits the Metastasis of Colorectal Cancer via Epidermal Growth Factor Receptor Signaling Axis.

Ginsenoside Rd is a saponin from ginseng and has been reported to have various biological activities. However, the effect of ginsenoside Rd on the metastasis of colorectal cancer (CRC) remains unknown. Here, we found that ginsenoside Rd decreased the colony-forming ability, migration, invasion, and wound-healing abilities of CRC cells, although it did not affect cell proliferation. In addition, using an inverse-docking assay, we found that ginsenoside Rd bound to epidermal growth factor receptor (EGFR) with a high binding affinity, inducing the downregulation of stemness- and epithelial-mesenchymal transition-related genes; these were partially rescued by either exogenous EGF treatment or ectopic expression of SOX2. Furthermore, ginsenoside Rd significantly decreased the number and size of tumor metastasis nodules in the livers, lungs, and kidneys of mouse model of metastasis. © 2018 IUBMB Life, 71(5):601-610, 2019.

Hedgehog Signaling in Cancer: A Prospective Therapeutic Target for Eradicating Cancer Stem Cells.

The Hedgehog (Hh) pathway is a signaling cascade that plays a crucial role in many fundamental processes, including embryonic development and tissue homeostasis. Moreover, emerging evidence has suggested that aberrant activation of Hh is associated with neoplastic transformations, malignant tumors, and drug resistance of a multitude of cancers. At the molecular level, it has been shown that Hh signaling drives the progression of cancers by regulating cancer cell proliferation, malignancy, metastasis, and the expansion of cancer stem cells (CSCs). Thus, a comprehensive understanding of Hh signaling during tumorigenesis and development of chemoresistance is necessary in order to identify potential therapeutic strategies to target various human cancers and their relapse. In this review, we discuss the molecular basis of the Hh signaling pathway and its abnormal activation in several types of human cancers. We also highlight the clinical development of Hh signaling inhibitors for cancer therapy as well as CSC-targeted therapy.

The anti-metastatic effect of ginsenoside Rb2 in colorectal cancer in an EGFR/SOX2-dependent manner.

Ginsenoside Rb2, a saponin from Panax ginseng, has been shown to have many functions. However, the effect of ginsenoside Rb2 on the metastasis of colorectal cancer (CRC) remains unknown. CRC cell lines HT29 and SW620 were used to determine the effects of ginsenoside Rb2 on the colony-forming, migration, invasion, and wound-healing abilities of CRC cells in vitro. Further, ginsenoside Rb2 was given intraperitoneally at 5 mg/kg of mouse body weight to check its effect on the metastasis of CRC cells in vivo. Ginsenoside Rb2 decreased colony-forming ability, migration, invasion, and wound healing of CRC cells in vitro, although it did not affect cell proliferation. As a possible mechanism, we found that ginsenoside Rb2 down-regulated the expression of stemness and Epithelial-mesenchymal transition (EMT)-related genes via the EGFR/SOX2 signaling axis; these were partially rescued by either exogenous EGF treatment or ectopic expression of SOX2. More importantly, ginsenoside Rb2 significantly reduced the number of metastatic nodules in the livers, lungs, and kidneys in a mouse model of metastasis. These results suggest that ginsenoside Rb2 could be used to treat the metastasis of CRC therapeutically or as a supplement.

Cancer Stem Cells (CSCs) in Drug Resistance and their Therapeutic Implications in Cancer Treatment.

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are suggested to be responsible for drug resistance and cancer relapse due in part to their ability to self-renew themselves and differentiate into heterogeneous lineages of cancer cells. Thus, it is important to understand the characteristics and mechanisms by which CSCs display resistance to therapeutic agents. In this review, we highlight the key features and mechanisms that regulate CSC function in drug resistance as well as recent breakthroughs of therapeutic approaches for targeting CSCs. This promises new insights of CSCs in drug resistance and provides better therapeutic rationales to accompany novel anticancer therapeutics.

Allogeneic Adipose-Derived Mesenchymal Stem Cell Transplantation Enhances the Expression of Angiogenic Factors in a Mouse Acute Hindlimb Ischemic Model.

Cell migration and molecular mechanisms during healing of damaged vascular or muscle tissues are emerging fields of interest worldwide. The study herein focuses on evaluating the role of allogenic adipose-derived mesenchymal stem cells (ADMSCs) in restoring damaged tissues. Using a hindlimb ischemic mouse model, ADMSC-mediated induction of cell migration and gene expression related to myocyte regeneration and angiogenesis were evaluated. ADMSCs were labeled with GFP (ADMSC-GFP). The proximal end of the femoral blood vessel of mice (over 6 months of age) are ligated at two positions then cut between the two ties. Hindlimb ischemic mice were randomly divided into two groups: Group I (n = 30) which was injected with PBS (100 µL) and Group II (n = 30) which was transplanted with ADMSC-GFP (106 cells/100 µL PBS) at the rectus femoris muscle. The migration of ADMSC-GFP in hindlimb was analyzed by UV-Vis system. The expression of genes related to angiogenesis and muscle tissue repair was quantified by real-time RT-PCR. The results showed that ADMSCs existed in the grafted hindlimb for 7 days. Grafted cells migrated to other damaged areas such as thigh and heel. In both groups the ischemic hindlimb showed an increased expression of several angiogenic genes, including Flt-1, Flk-1, and Ang-2. In particular, the expression of Ang-2 and myogenic-related gene MyoD was significantly increased in the ADMSC-treated group compared to the PBS-treated (control) group; the expression increased at day 28 compared to day 3. The other factors, such as VE-Cadherin, HGF, CD31, Myf5, and TGF-ß, were also more highly expressed in the ADMSC-treated group than in the control group. Thus, grafted ADMSCs were able to migrate to other areas in the injured hindlimb, persist for approximately 7 days, and have a significantly positive impact on stimulating expression of myogenic- and angiogenesis-related genes.

Extracellular vesicles of ETV2 transfected fibroblasts stimulate endothelial cells and improve neovascularization in a murine model of hindlimb ischemia.

Ischemia are common conditions related to lack of blood supply to tissues. Depending on the ischemic sites, ischemia can cause different diseases, such as hindlimb ischemia, heart infarction and stroke. This study aims to evaluate how extracellular vesicles (EVs) derived from ETV2 transfected fibroblasts affect endothelial cell proliferation and neovascularization in a murine model of hindlimb ischemia. Human fibroblasts were isolated and cultured under standard conditions and expanded to the 3th passage before use in experiments. Human fibroblasts were transduced with a viral vector containing the ETV2 gene. Transduced cells were selected by puromycin treatment. These cells were further cultured for collection of EVs, which were isolated from culture supernatant. Following co-culture with endothelial cells, EVs were evaluated for their effect on endothelial cell proliferation and were directly injected into ischemic tissues of a murine model of hindlimb ischemia. The results showed that EVs could induce endothelial cell proliferation in vitro and improved neovascularization in a murine model of hindlimb ischemia. Our results suggest that EVs derived from ETV2-transfected fibroblasts can be promising non-cellular products for the regeneration of blood vessels.

ETV-2 activated proliferation of endothelial cells and attenuated acute hindlimb ischemia in mice.

Ischemia is the reduction of blood flow to tissues by injury of blood vessels. Depending on the sites of tissues and grade of ischemia, ischemia can cause many serious complications. This study aimed to evaluate the effects of the E-twenty six (ETS) factor Ets variant 2 (ETV2) gene expression in angiogenesis and the effect of ETV2 gene therapy in a mouse model of hindlimb ischemia. The role of ETV2 on endothelial cell proliferation was evaluated in vitro. Knockdown of ETV2 expression was done using short hairpin RNA (shRNA) lentiviral viral particles. The ETV2 viral vector was injected into the skeletal muscles at the ligated and burned sites of the hindlimb and evaluated for its efficacy as a gene therapy modality for ischemia. Vascular regeneration in mice was indirectly evaluated by changes in mouse survival, necrotic grades of the leg, normal blood oxygen saturation level (SpO2), and blood flow by trypan blue injection assay. Preliminary data showed that ETV2 expression played a role in angiogenesis of endothelial cells. ETV2 overexpression could trigger and stimulate proliferation of skeletal endothelial cells. In vivo knockdown of ETV2 expression inhibited the auto-recovery of ischemic hindlimb, while overexpression of ETV2 helped to rescue leg loss and reduce necrosis, significantly improving angiogenesis in hindlimb ischemia. Our findings demonstrate that ETV2 gene therapy is a potentially effective modality for vascular regeneration.

Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages.

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

Interferon-induced transmembrane protein 1 (IFITM1) is required for the progression of colorectal cancer.

Interferon-induced transmembrane protein 1 (IFITM1) has been shown to be implicated in multiple cancers, yet little is known about biological significance of IFITM1 in colorectal cancer. Here, we show that IFITM1 is highly expressed in metastatic colorectal cancer cell lines as well as colorectal patient-derived tumor samples, and its expression is associated with a poor prognosis of the disease. Also, IFITM1 depletion resulted in a significant reduction in the mobility of cancer cell lines, whereas ectopic expression of IFITM1 promoted the migration of cancer cells. Epithelial-mesenchymal transition (EMT) signature was dysregulated by both loss and gain of function of IFITM1, which was partially reverted by Caveolin-1 (CAV1). Therefore, these results suggest that IFITM1 may be a prognostic marker and an attractive target to achieve better therapeutic outcomes in colorectal cancer.