The therapeutic potential of mesenchymal stem cells in lung cancer: benefits, risks and challenges Abstract Background Lung cancer is one of the most challenging diseases to treat. In the past decades standard therapy including surgery, chemo- and radiation therapy, alone or in combination has not changed the high mortality rate and poor prognosis. In recent years, mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumor tropism, anti-tumor and immunoregulatory properties. MSCs release biomolecules that are thought to exert the same beneficial effects as their cellular counterparts and, as such, they may offer practical possibilities of using MSC-secreted products. Owing to their innate affinity to home to tumor sites, MSCs have also gained interest as selective vehicles for the delivery of anti-cancer agents. However, MSCs are also known to confer pro-oncogenic effects, rendering them into double-sword weapons against neoplastic diseases. Conclusions Here, we present published data on the cell- and secretome-based therapeutic competences of MSCs, as well as on their potential as engineered delivery vectors for the treatment of lung cancer. Despite the controversial role of MSCs in the context of lung cancer therapy, current findings support hopeful perspectives to harness the potential of MSC-based regimens that may augment current treatment modalities in lung cancer.

Human bone marrow-derived mesenchymal stem cell-secreted exosomes overexpressing microRNA-34a ameliorate glioblastoma development via down-regulating MYCN Abstract Purpose Exosomes play important roles in intercellular communication through signaling pathways affecting tumor microenvironment modulation and tumor proliferation, including those in glioblastoma (GBM). As yet, however, limited studies have been conducted on the inhibitory effect of human bone marrow-derived mesenchymal stem cell (hBMSC)-derived exosomes on GBM development. Therefore, we set out to assess the role of hBMSC secreted exosomes, in particular those carrying microRNA-34a (miR-34a), in the development of GBM. Methods Microarray-based expression analysis was employed to identify differentially expressed genes and to predict miRNAs regulating MYCN expression. Next, hBMSCs were transfected with a miR-34a mimic or inhibitor after which exosomes were isolated. Proliferation, apoptosis, migration, invasion and temozolomide (TMZ) chemosensitivity of exosome-exposed GBM cells (T-98G, LN229 and A-172) were measured in vitro. The mechanism underlying MYCN regulation was investigated using lentiviral transfections. The in vivo inhibitory effect of exosomal miR-34a was measured in nude mice xenografted with GBM cells through subcutaneous injection of hBMSCs with an upregulated miR34a content. Results We found that poorly-expressed miR-34a specifically targeted and negatively regulated the expression of MYCN in GBM cells. In addition we found that miR-34a was delivered to T-98G, LN229 and A-172 GBM cells via hBMSC-derived exosomes. Exogenous overexpression of miR-34a in hBMSC-derived exosomes resulted in inhibition of GBM cell proliferation, invasion, migration and tumorigenesis in vitro and in vivo, while promoting the chemosensitivity of GBM cells to TMZ by silencing MYCN. Conclusions From our data we conclude that hBMSC-derived exosomes overexpressing miR-34a may be instrumental for the therapeutic targeting and clinical management of GBM.

The emerging role of noncoding RNAs in colorectal cancer chemoresistance Abstract Background Colorectal cancer (CRC) is the third most prevalent cancer in the world and one of the most lethal human malignancies. Chemotherapy with 5-fluorouracil, platinum, hydroxycamptothecin, vincristine, methotrexate, irinotecan, paclitaxel and/or cetuximab has significantly improved the survival of CRC patients. However, most CRC patients eventually develop chemoresistance, resulting in a poor prognosis. The mechanisms involved in CRC chemoresistance are complex and, as yet, inconclusive. Noncoding RNAs (ncRNAs), such as small nucleolar RNAs (snoRNAs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), represent transcripts without protein-coding potential. Accumulating evidence indicates that multiple deregulated ncRNAs, including miRNAs and lncRNAs, play pivotal roles in the development of chemoresistance in CRC. This notion has potential clinical implications. Conclusions In this review, we highlight the emerging roles and the regulatory mechanisms by which miRNAs and lncRNAs affect CRC chemoresistance. Tumor-specific miRNAs and lncRNAs may serve as novel therapeutic targets and prognostic biomarkers for CRC.

Phenethyl isothiocyanate hampers growth and progression of HER2-positive breast and ovarian carcinoma by targeting their stem cell compartment Abstract Purpose Isothiocyanates elicit anticancer effects by targeting cancer stem cells (CSCs). Here, we tested the antitumor activity of phenethyl-isothiocyanate (PEITC), either alone or in combination with trastuzumab, in HER2-positive tumor models. Methods We assessed the in vitro anticancer activity of PEITC, alone or combined with trastuzumab, in HER2-positive BT474, SKBR3, HCC1954 and SKOV3 cancer cells by measuring their sphere forming efficiency (SFE). The expression of the human/rodent CSC biomarkers aldehyde-dehydrogenase (ALDH) and CD29High/CD24+/Sca1Low was evaluated by cytofluorimetric analysis. The expression of wild type HER2 (WTHER2), its splice variant d16HER2 and NOTCH was analysed by quantitative RT-PCR and Western blotting. The in vivo activity of PEITC and trastuzumab was evaluated in mice orthotopically implanted with MI6 tumor cells transgenic for the human d16HER2 splice isoform. Magnetic resonance imaging/spectroscopy and immunohistochemistry were used to assess morpho-functional and metabolic profiles of treated versus untreated mice. Results We found that PEITC significantly impaired the SFE of HER2-positive human cancer cells by decreasing their ALDH-positive compartments. The anti-CSC activity of PEITC was demonstrated by a reduced expression/activation of established cancer-stemness biomarkers. Similar results were obtained with MI6 cells, where PEITC, alone or in combination with trastuzumab, significantly inhibited their SFE. We also found that PEITC hampered the in vivo growth of MI6 nodules by inducing hemorrhagic and necrotic intra-tumor areas and, in combination with trastuzumab, by significantly reducing spontaneous tumor development in d16HER2 transgenic mice. Conclusions Our results indicate that PEITC targets HER2-positive CSCs and that its combination with trastuzumab may pave the way for a novel therapeutic strategy for HER2-positive tumors.

Piperlongumine potentiates the antitumor efficacy of oxaliplatin through ROS induction in gastric cancer cells Abstract Purpose Oxaliplatin is one of the most commonly used chemotherapeutic agents in the treatment of various cancers, including gastric cancer. It has, however, a narrow therapeutic index due to its toxicity and the occurrence of drug resistance. Therefore, there is a pressing need to develop novel therapies to potentiate the efficacy and reduce the toxicity of oxaliplatin. Piperlongumine (PL), an alkaloid isolated from Piper longum L., has recently been identified as a potent agent against cancer cells in vitro and in vivo. In the present study, we investigated whether PL can potentiate the antitumor effect of oxaliplatin in gastric cancer cells. Methods Cellular apoptosis and ROS levels were analyzed by flow cytometry. Thioredoxin reductase 1 (TrxR1) activity in gastric cancer cells or tumor tissues was determined using an endpoint insulin reduction assay. Western blotting was used to analyze the expression levels of the indicated proteins. Nude mice xenograft models were used to test the effects of PL and oxaliplatin combinations on gastric cancer cell growth in vivo. Results We found that PL significantly enhanced oxaliplatin-induced growth inhibition in both gastric and colon cancer cells. Moreover, we found that PL potentiated the antitumor effect of oxaliplatin by inhibiting TrxR1 activity. PL combined with oxaliplatin markedly suppressed the activity of TrxR1, resulting in the accumulation of ROS and, thereby, DNA damage induction and p38 and JNK signaling pathway activation. Pretreatment with antioxidant N-acetyl-L-cysteine (NAC) significantly abrogated the combined treatment-induced ROS generation, DNA damage and apoptosis. Importantly, we found that activation of the p38 and JNK signaling pathways prompted by PL and oxaliplatin was also reversed by NAC pretreatment. In vivo, we found that PL combined with oxaliplatin significantly suppressed tumor growth in a gastric cancer xenograft model, and effectively reduced the activity of TrxR1 in tumor tissues. Remarkably, we found that PL attenuated body weight loss evoked by oxaliplatin treatment. Conclusions Our data support a synergistic effect of PL and oxaliplatin and suggest that application of its combination may be more effective for the treatment of gastric cancer than oxaliplatin alone.

Peroxiredoxin 4 suppresses anoikis and augments growth and metastasis of hepatocellular carcinoma cells through the β-catenin/ID2 pathway Abstract Purpose Peroxiredoxin 4 (PRDX4) has been reported to play a dual role in the progression of hepatocellular carcinoma (HCC). As yet, however, the underlying molecular mechanism has not been fully elucidated. Methods We examined the effects of PRDX4 on the growth and survival of HCC cells in an anchorage-independent microenvironment. The regulation of β-catenin stability and activity by PRDX4 was investigated. Results We found that PRDX4 depletion reduced, and PRDX4 overexpression increased, both anchorage-dependent and anchorage-independent growth of HCC cells. We also found that PRDX4 depletion caused an overproduction of reactive oxygen species (ROS) in HCC cells, especially under suspension conditions. PRDX4 knockdown predisposed HCC cells to anoikis, whereas PRDX4 overexpression induced resistance to anoikis. Subsequent in vivo studies confirmed that PRDX4 deficiency blocks HCC tumor growth and pulmonary metastasis. Mechanistically, we found that RDX4 reduced β-TrCP-mediated β-catenin ubiquitination and enhanced β-catenin protein stability, consequently leading to activation of β-catenin signaling. Silencing of β-catenin impaired PRDX4-mediated anchorage-independent growth and survival, whereas β-catenin overexpression increased the survival and growth of PRDX4-depleted cells under anchorage-independent conditions. Further investigation revealed that the β-catenin downstream gene ID2 is responsible for the oncogenic activity of PRDX4 in HCC cells, promoting anchorage-independent growth and anoikis resistance. Conclusions PRDX4 reduces anoikis and promotes tumorigenesis and metastasis of HCC cells through stabilization of the β-catenin protein and upregulation of ID2. Targeting of PRDX4 may represent a promising strategy to block HCC cell growth and metastasis.

The tetraspanin CD81 mediates the growth and metastases of human osteosarcoma Abstract Purpose CD81 is a member of the tetraspanin family of membrane proteins. Recently, it has been shown that CD81 may be involved in cancer cell proliferation and metastasis. As yet, however, there have been few reports on the expression and role of CD81 in osteosarcoma. Methods The expression of CD81 was investigated in human osteoblast cell line hFOB1.19 and in human osteosarcoma cell lines Saos2, MG63 and 143B. The expression of CD81 was inhibited in osteosarcoma cells using siRNA after which cell proliferation, migration and invasion were assessed. We also used Western blotting to investigate the phosphorylation status of Akt, Erk, JNK and p38, and measured the expression of MMP-2, MMP-9 and MT1-MMP. In addition, we used a CRISPR/Cas9 system to stably knock out CD81 expression in 143B cells, transplanted the cells into mice, and assessed tumor formation and lung metastasis in these mice compared to those in the control group. Results We found that CD81 was expressed in the human osteoblast cell line and in all osteosarcoma cell lines tested. The osteosarcoma cell line 143B exhibited a particularly high level of expression. In addition, we found that osteosarcoma cell proliferation, migration and invasion were decreased after CD81 inhibition, and that the phosphorylation of Akt and Erk was suppressed. Also, the expression levels of MMP-2, MMP-9 and MT1-MMP were found to be suppressed, with MMP-9 showing the greatest suppression. In vivo, we found that mice transplanted with CD81 knockout 143B cells exhibited significantly less tumor formation and lung metastasis than mice in the control group. Conclusion Based on our findings we conclude that inhibition of CD81 suppresses intracellular signaling and reduces tumorigenesis and lung metastasis in osteosarcoma cells.

Epigenetics of lung cancer: a translational perspective Abstract Background Lung cancer remains the most common cause of cancer-related death, with a 5-year survival rate of only 18%. In recent years, the development of targeted pharmacological agents and immunotherapies has substantially increased the survival of a subset of patients. However, most patients lack such efficacious therapy and are, thus, treated with classical chemotherapy with poor clinical outcomes. Therefore, novel therapeutic strategies are urgently needed. In recent years, the development of epigenetic assays and their application to cancer research have highlighted the relevance of epigenetic regulation in the initiation, development, progression and treatment of lung cancer. Conclusions A variety of epigenetic modifications do occur at different steps of lung cancer development, some of which are key to tumor progression. The rise of cutting-edge technologies such as single cell epigenomics is, and will continue to be, crucial for uncovering epigenetic events at a single cell resolution, leading to a better understanding of the biology underlying lung cancer development and to the design of novel therapeutic options. This approach has already led to the development of strategies involving single agents or combined agents targeting epigenetic modifiers, currently in clinical trials. Here, we will discuss the epigenetics of every step of lung cancer development, as well as the translation of these findings into clinical applications.

QKI deficiency maintains glioma stem cell stemness by activating the SHH/GLI1 signaling pathway Abstract Purpose Glioblastoma (GBM) stem cells (GSCs) have been found to be the main cause of malignant GBM progression. It has also been found that Quaking homolog (QKI) plays a predominant role in driving GBM development. Here, we aimed to asses the role of QKI in maintaining GSC stemness and inducing the invasiveness of GBM cells. Methods Public databases were used to assess the expression of QKI and its correlation with stemness markers in primary GBMs. The CRISPR-Cas9 technology was used to generate QKI knockout GBM cells, and RNA immunoprecipitation was used to assess QKI-GLI1 protein-mRNA interactions. In addition, in vitro and in vivo GBM cell proliferation, migration, xenografting and neurosphere formation assays were performed. Results Using public GBM databases, QKI was identified as a potential GSC regulator. We found that QKI could inhibit stem-like cell (SLC) stemness and prolong the survival of xenografted mice. Mechanistically, we found that QKI knockout increased the GLI Family Zinc Finger 1 (GLI1) mRNA level, which is essential for maintaining the self-renewal ability of GSCs. In addition, we found that QKI knockout activated the Hedgehog signaling pathway via Tra-2 and GLI response element (TGE)-specific GLI1 mRNA disruption. Conclusion Our data indicate that upregulation of GLI1 induced by QKI deficiency maintains GSC stemness and enhances the invasiveness of GBM cells, thereby hinting at new options for the treatment of GBM.

Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation Abstract Purpose The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia. Methods Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4–11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2′-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element. Results We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser588 of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4. Conclusions From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.