BTB and CNC homology 1 (Bach1) induces lung cancer stem cell phenotypes by stimulating CD44 expression

Background Growing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently. Methods The expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67. Results The ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44− CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression. Conclusion In summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

Activating transcription factor 5 (ATF5) promotes tumorigenic capability and activates the Wnt/b-catenin pathway in bladder cancer

Background In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. Methods Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. Results ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/β-catenin pathway. Conclusions This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

TGF-β1 Induced Deficiency of Linc00261 Promotes Epithelial-mesenchymal-transition and Stemness of Hepatocellular Carcinoma via Modulating SMAD3

Emerging evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the metastasis and recurrence of hepatocellular carcinoma (HCC).Kinds of lncRNAs were found to be involved in regulating epithelial-mesenchymal transition (EMT) or stem-like traits in human cancers, however, the molecular mechanism and signaling pathways targeting EMT and stemness remains largely unknown. Previously, we found that linc00261 was down-regulated in HCC and associated with multiple worse clinic pathological parameters and poor prognosis. Here, we show that linc00261 was down-regulated in TGF-β1 stimulated cells, and forced expression of linc00261 attenuated EMT and stem-like traits in HCC.Linc00261 also inhibited the tumor sphere forming in vitro and decreased the tumorigenicity in vivo. Furthermore, we revealed that linc00261 suppressed the expression and phosphorylation of SMAD3 (p-SMAD3), which is a core transcriptional modulator in TGF-β1 signaling mediated EMT and the acquisition of stemness traits. A negative correlation between linc00261 and p-SMAD3 was determined in HCC samples.Conclusion: Our study revealed that linc00261suppressed EMT and stem-like traits of HCC cells by inhibiting TGF-β1/SMAD3 signaling.

Disulfiram Sensitizes a Therapeutic-Resistant Glioblastoma to the TGF-β Receptor Inhibitor

Despite neurosurgery following radiation and chemotherapy, residual glioblastoma (GBM) cells develop therapeutic resistance (TR) leading to recurrence. The GBM heterogeneity confers TR. Therefore, an effective strategy must target cancer stem cells (CSCs) and other malignant cancer cells. TGF-β and mesenchymal transition are the indicators for poor prognoses. The activity of aldehyde dehydrogenases (ALDHs) is a functional CSC marker. However, the interplay between TGF-β and ALDHs remains unclear. We developed radiation-resistant and radiation-temozolomide-resistant GBM models to investigate the underlying mechanisms conferring TR. Galunisertib is a drug targeting TGF-β receptors. Disulfiram (DSF) is an anti-alcoholism drug which functions by inhibiting ALDHs. The anti-tumor effects of combining DSF and Galunisertib were evaluated by in vitro cell grow, wound healing, Transwell assays, and in vivo orthotopic GBM model. Mesenchymal-like phenotype was facilitated by TGF-β in TR GBM. Additionally, TR activated ALDHs. DSF inhibited TR-induced cell migration and tumor sphere formation. However, DSF did not affect the tumor growth in vivo. Spectacularly, DSF sensitized TR GBM to Galunisertib both in vitro and in vivo. ALDH activity positively correlated with TGF-β-induced mesenchymal properties in TR GBM. CSCs and mesenchymal-like GBM cells targeted together by combining DSF and Galunisertib may be a good therapeutic strategy for recurrent GBM patients.

Activating Transcription Factor 5 (ATF5) Promotes Tumorigenic Capability and Activates the Wnt/b-Catenin Pathway in Bladder Cancer

Background In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. Methods Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. Results ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/β-catenin pathway. Conclusions This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

Specific and Aspecific Molecular Checkpoints as Potential Targets for Dismantling Tumor Hierarchy and Preventing Relapse and Metastasis Through Shielded Cytolytic Treatments

I have recently theorized that several similarities exist between the tumor process and embryo development. Starting from an initial cancer stem cell (CSC0), similar to an embryonic stem cell (ESC), after implantation in a niche, primary self-renewing CSCs (CSC1s) would arise, which then generate secondary proliferating CSCs (CSC2s). From these epithelial CSCs, tertiary mesenchymal CSCs (CSC3s) would arise, which, under favorable stereotrophic conditions, by asymmetric proliferation, would generate cancer progenitor cells (CPCs) and then cancer differentiated cells (CDCs), thus giving a defined cell heterogeneity and hierarchy. CSC1s–CSC2s–CSC3s–CPCs–CDCs would constitute a defined “tumor growth module,” able to generate new tumor modules, forming a spherical avascular mass, similar to a tumor sphere. Further growth in situ of this initial tumor would require implantation in the host and vascularization through the overexpression of some aspecific checkpoint molecules, such as CD44, ID, LIF, HSP70, and HLA-G. To expand and spread in the host tissues, this vascularized tumor would then carry on a real growth strategy based on other specific checkpoint factors, such as those contained in the extracellular vesicles (EVs), namely, microRNAs, messenger RNAs, long non-coding RNAs, and integrins. These EV components would be crucial in tumor progression because they can mediate intercellular communications in the surrounding microenvironment and systemically, dictating to recipient cells a new tumor-enslaved phenotype, thus determining pre-metastatic conditions. Moreover, by their induction properties, the EV contents could also frustrate in time the effects of cytolytic tumor therapies, where EVs released by killed CSCs might enter other cancer and non-cancer cells, thus giving chemoresistance, non-CSC/CSC transition (recurrence), and metastasis. Thus, antitumor cytotoxic treatments, “shielded” from the EV-specific checkpoints by suitable adjuvant agents, simultaneously targeting the aforesaid aspecific checkpoints should be necessary for dismantling the hierarchic tumor structure, avoiding recurrence and preventing metastasis.

A Regulatory Loop Involving Notch and Wnt Signaling Maintains Leukemia Stem Cells in T-Cell Acute Lymphoblastic Leukemia

Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that β-catenin is over-expressed in almost all T-ALL patients and flow sorted β-cateninhigh fractions are highly resistant to therapy, leading to liver metastases in nude mice as well as dysregulated lncRNAs. Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of β-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from β-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with β-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to β-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.

EMBR-12. TARGETING THE RNA-BINDING PROTEIN LIN28B IN GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. We have previously demonstrated an oncogenic role for the RNA-binding protein (RBP) LIN28B in neuroblastoma. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28A/B and other oncogenes. LIN28B has also been found to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. LIN28B levels are highest in Group 3 MB patients, and its overexpression is associated with significantly worse survival. Here we demonstrate that down-regulation of LIN28B using shRNA results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation The LIN28 inhibitor 1632 also leads to significant reduction in G3 MB cell proliferation. In addition, we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally RNA-seq profiling following LIN28B depletion reveals additional components of the LIN28B pathway which may be amenable to therapeutic targeting. This work will help define the role for LIN28B in Group 3 MB aggressiveness and establish LIN28B and LIN28B-driven networks as novel therapeutic targets in these patients.

Multilevel regulation of Wnt signaling by Zic2 in colon cancer due to mutation of β-catenin

Zinc-finger of the cerebellum 2 (Zic2) is widely implicated in cancers, but the role of Zic2 in tumorigenesis is bilateral. A recent study indicated that Zic2 could render colon cancer cells more resistant to low glucose-induced apoptosis. However, the functional roles of Zic2 in colon cancer and the underlying molecular mechanism remain elusive. Herein, we demonstrated that Zic2 was highly expressed in colon cancer tissues and correlated with poor survival. Knockdown of Zic2 inhibited colon cancer cell growth, arrested the cell cycle transition from G0/G1 to S phase, and suppressed tumor sphere formation in vitro; in addition, silencing Zic2 retarded xenograft tumor formation in vivo. Consistently, ectopic expression of Zic2 had the opposite effects. Mechanistically, Zic2 executed its oncogenic role in colon cancer by enhancing Wnt/β-catenin signaling. Zic2 directly binds to the promoter of Axin2 and transcriptionally represses Axin2 expression and subsequently promotes the accumulation and nuclear translocation of β-catenin. Meanwhile, Zic2 could activate Wnt signaling by interacting with β-catenin. Intriguingly, in HCT116 cells with intrinsic Ser45 mutation of β-catenin, which blocks the degradation-related phosphorylation of β-catenin by CK1, modified Zic2 expression did not affect the protein level of β-catenin. Altogether, our findings uncover a novel multilevel mechanism for the oncogenic activity of Zic2 in colon cancer and suggest Zic2 as a potential therapeutic target for colon cancer patients.

Androgen Receptor, Although Not a Specific Marker For, Is a Novel Target to Suppress Glioma Stem Cells as a Therapeutic Strategy for Glioblastoma

Targeting androgen receptor (AR) has been shown to be promising in treating glioblastoma (GBM) in cell culture and flank implant models but the mechanisms remain unclear. AR antagonists including enzalutamide are available for treating prostate cancer patients in clinic and can pass the blood–brain barrier, thus are potentially good candidates for GBM treatment but have not been tested in GBM orthotopically. Our current studies confirmed that in patients, a majority of GBM tumors overexpress AR in both genders. Enzalutamide inhibited the proliferation of GBM cells both in vitro and in vivo. Although confocal microscopy demonstrated that AR is expressed but not specifically in glioma cancer stem cells (CSCs) (CD133+), enzalutamide treatment significantly decreased CSC population in cultured monolayer cells and spheroids, suppressed tumor sphere-forming capacity of GBM cells, and downregulated CSC gene expression at mRNA and protein levels in a dose- and time-dependent manner. We have, for the first time, demonstrated that enzalutamide treatment decreased the density of CSCs in vivo and improved survival in an orthotopic GBM mouse model. We conclude that AR antagonists potently target glioma CSCs in addition to suppressing the overall proliferation of GBM cells as a mechanism supporting their repurposing for clinical applications treating GBM.