Promotes Cancer Stem Research Articles

Interaction of lncRNA-MALAT1 and miR-124 regulates HBx-induced cancer stem cell properties in HepG2 through PI3K/Akt signaling.

Hepatitis B virus X protein (HBx) plays a crucial role in initiating and promoting HBV-induced hepatocellular carcinoma (HCC) development. Reports indicated that HBx promotes cancer stem cell (CSC) generation, which may be associated with HBV-related HCC. Noncoding RNA miR-124 and long noncoding RNA (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) were considered to be involved deeply in the progress of HBx-related HCC. Hence, the underlying mechanism of miR-124 and lncRNA-MALAT1 in regulating HBx-promoted CSC needs to be studied. In present study, HepG2-X cell line was induced by transfect HBx into HepG2 cells. Overexpressing of miR-124 or silencing of lncRNA-MALAT1 was completed by transfecting miR-124 mimic or shMALAT1 into HepG2-X cells. HBx-induced CSC properties and tumorigenic potential of HepG2 cells were determined by detecting CSC marker expression, colony formation assay, and xenograft tumorigenesis. The mechanism of HBx-induced CSC properties was explored by PI3K/Akt inhibitor. Interaction of miR-124 and lncRNA-MALAT1 was detected by luciferase reporter assay. HBx promoted CSC properties through upregulating stemness markers and reprogramming proteins, and contributed to tumorigenicity of HepG2-X cells both in vivo and in vitro. Inhibition of Akt activation blocked the HBx-stimulated reprogramming proteins and stemness markers. HBx upregulated lncRNA-MALAT1 expression while downregulating miR-124 expression in HepG2-X cells. miR-124 interacts with lncRNA-MALAT1 by direct targeting. Overexpression of miR-124 or silencing of lncRNA-MALAT1 both blocked HBx-induced CSC generation, stemness-related factor activation and tumorigenicity via PI3K/Akt signaling. Our results demonstrated that miR-124 interact with lncRNA-MALAT1 and involve in regulating HBx-induced CSC properties via PI3K/Akt signaling.

Transfection of T-Box Transcription Factor BRACHYURY and SOX2 Synergistically Promote Self-Renewal and Invasive Phenotype in Oral Cancer Cells.

Recent studies suggest that epithelial–mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-β2. In particular, the effects on FIBRONECTIN and TGF-β2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.

RETRACTED ARTICLE: Periostin secreted by cancer-associated fibroblasts promotes cancer stemness in head and neck cancer by activating protein tyrosine kinase 7

Protein tyrosine kinase 7 (PTK7) and cancer-associated fibroblasts (CAFs) play important roles in cancer stemness, respectively. However, little is known about interaction between CAFs and PTK7 in cancers. In this study, we showed that PTK7 was significantly correlated with the Wnt/β-Catenin pathway and aggressive clinicopathologic features in human head and neck squamous cell carcinoma (HNSCC). Meanwhile, animal experiments showed that PTK7 enhanced chemoresistance and lung metastasis of HNSCC in vivo. In addition, co-immunoprecipitation (co-IP) assay demonstrated that POSTN secreted by CAFs was a potential upstream ligand of PTK7 which might act as a receptor. Further analysis revealed that POSTN promoted the cancer stem cell (CSC)-like phenotype via PTK7–Wnt/β-Catenin signaling, including the proliferation and invasion of HNSCC cells in vitro, as well as tumor initiation and progression in vivo. Collectively, our study proved that CAF-derived POSTN might promote cancer stemness via interacting with PTK7 in HNSCC, suggesting that the combination of POSTN and PTK7 might be a potential prognostic and diagnostic indicator and a promising therapeutic target.

GLI1 promotes cancer stemness through intracellular signaling pathway PI3K/Akt/NFκB in colorectal adenocarcinoma

The role of Hedgehog (HH)/ glioma-associated oncogene homolog 1 (GLI1) pathway has been implicated in a variety of cancer entities, and the targeted pathway inhibition mediated by GLI1 is of therapeutic relevance. However, its oncogenicity and cross-talks with other cancer pathways including PI3K/Akt/NFκB, which modulates the HH/GLI1 signal strength, have rarely been explored in colorectal adenocarcinoma. We assessed the expression of GLI1 and its relationship with other cancer stemness genes, cell cycle markers, epithelial-mesenchymal transition (EMT), PI3K/Akt/NFκB signaling pathway genes, and HIF1α in 100 paraffin-embedded colorectal adenocarcinoma tissue samples using immunohistochemistry. We further addressed the effect of GLI1 on EMT, cell cycle, and its putative interaction with the PI3K/Akt/NFκB cascade in colorectal adenocarcinoma cell lines. The expression of GLI1 in colorectal adenocarcinoma tissues was found to correlate with the clinical stages, and distant metastasis. Moreover, GLI1 was found to be an independent predictor of poor overall survival and disease-free survival in colorectal adenocarcinoma. GLI1-expressing cancer cells also expressed their representative cancer stem-like cell (CSC) markers (SOX9 and CD133), as well as HIF1α. GLI1 expression was also strongly linked to EMT-related and PI3K/Akt/NFκB signaling genes. Downregulation of GLI1 by inhibitor treatment in colorectal adenocarcinoma cell lines resulted in reduced expression of CSC markers, cell clonogenicity, S-phase subpopulations, as well as the migration and invasion ability. Importantly, Akt inhibitor Perifosine significantly inhibited the expression of pAkt and GLI1 in colorectal adenocarcinoma cells. Combination of GLI1 inhibitor GANT61 and NFκB p65 inhibitor QZN exhibited much higher inhibition compared to using any of them individually on colorectal adenocarcinoma cells. We suggested that GLI1 may be a novel stem cell marker, and cancer stemness was activated via PI3K/Akt/NFκB pathway. In addition, co-targeting GLI1 and PI3K/Akt/NFκB signaling simultaneously might provide an alternative therapeutic strategy for colorectal adenocarcinoma patients.

Non-Coding RNA Pvt1 Promotes Cancer Stem Cell-Like Traits in Nasopharyngeal Cancer via Inhibiting miR-1207.

Nasopharyngeal carcinoma (NPC) is a kind of head-neck malignant tumor. lncRNA-PVT1 can promote the proliferation of carcinoma cells, and induce cells to have stem cell-like potentials. However, the function of PVT1 in NPC cells is not clear. The expressions of lncRNA-PVT1 and the expressions of the stem cell markers in NPC tissues or cell lines were investigated by qRT-PCR or western blot. The cell proliferation, and the ability of NPC cells to form spherical, clonal colonies were investigated by MTT assay, colony formation assay, and tumor-sphere formation assay. Cancer stem cells surface markers were detected by flow cytometry and western blot. PI3K/AKT signal activation in NPC cells was determined by western blot. PVT1 was significantly up-regulated in both NPC tissues and cell lines and associated with poor prognosis. PVT1 knockdown reduced NPC cells viability, clonogenicity, the cell surface CD44+/CD24- stem phenotype, and the expressions of the stem cell markers in NPC cells, including Oct4, c-Myc, SOX2, and ALDH. Furthermore, PVT1 negatively regulates the expression levels of miR-1207 in NPC cells and spheres cells, which is critical for NPC stemness. Knockdown of miR-1207 promoted stem phenotype and the expressions of the stem cell markers in NPC cells. Moreover, phosphor-PI3K (p-PI3K) and phosphor-AKT (p-AKT) were found to be down-regulated after PVT1 siRNAs transfection in NPC cells. And miR-1207 inhibitor transfection reversed the all the effects brought by PVT1 knockdown. Pvt1 promotes cancer stem cell-like properties in NPC cells via inhibiting miR-1207 and activating the PI3K/AKT signal pathway.

Abstract 5226: Tiam1 is a key modulator of chemoresistance in colorectal cancer

Abstract Purpose: Emerging evidence indicates that a small subpopulation of cancer cells with stem cell-like properties are responsible for acquisition of drug resistance in colorectal cancer (CRC). Intriguingly, the acquisition of such a stem-like state and subsequent drug resistance has been shown to be induced by the tumor microenvironment. In particular, cancer associated fibroblasts (CAFs) have been identified as a key component of tumor microenvironment which promote cancer stemness. Wnt signaling is a well-recognized pathway that drives self-renewal, and recently one of the Wnt-responsive genes, Tiam1, a guanine nucleotide exchange factor specific for Rac1, was shown to be overexpressed in CRC and associated with an aggressive cancer phenotype. Herein, we have investigated Tiam1 as a key modulator of chemoresistance in CRC. Methods: Tiam1 expression was assessed in resected CRC tissues from 300 patients who did or did not respond to chemotherapy. We used siRNA and CRISPR/CAS9 approach in three cell lines and animal models to examine whether inhibition of Tiam1 affects chemosensitivity in CRC. In addition, we established CRC patient derived-CAFs and examined whether the drug sensitivity of CRC cells was altered by supplementation of CAF-derived conditioned medium. Results: High Tiam1 expression significantly correlated with poor prognosis in CRC patients (P=0.032), and emerged as an independent prognostic factor (HR 5.06, 95% CI 1.10-23.2, P=0.038). In addition, Tiam1 overexpression is associated with patients who did not respond to chemotherapy (P=0.0005). We demonstrated that siRNA-mediated inhibition of Tiam1 enhanced sensitivity to three chemotherapeutic drugs (5-fluorouracil, oxaliplatin and irinotecan), and reduced tumor invasiveness in HCT116, SW480 and SW620 cell lines. Silencing of Tiam1 resulted in downregulation of stemness-related genes and spheroid formation ability. Furthermore, we validated that Tiam1-knockdown enhanced drug sensitivity and decreased the expression of stemness-related genes in xenograft tumors. Finally, we illustrated that the conditioned medium (CM) derived from CAFs led to increased stemness and chemoresistance in CRC cells through Tiam1 overexpression, and silencing of Tiam1 in CAFs resulted in reversal of stemness and chemoresistance properties. From a mechanistic standpoint, the target genes of Wnt signaling were also upregulated during co-culture with CM in CRC cells. Conclusion: We have firstly demonstrated that Tiam1 is overexpressed in CRC patients who did not respond to chemotherapeutic drugs; and furthermore, high Tiam1 expression emerged as an independent prognostic factor in CRC. Mechanistically, CAFs enhanced CRC chemoresistance through Tiam1 overexpression. Collectively, these results suggest that Tiam1 suppression in stroma may sensitize CRC cells to chemotherapeutic agents, suggesting that Tiam1 may be an attractive therapeutic target in colorectal cancer. Citation Format: Daisuke Izumi. Tiam1 is a key modulator of chemoresistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5226.

Abstract 532: PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models

Abstract Proline, glutamic acid, and leucine rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, we sought to identify interacting partners unique to cytoplasmic PELP1 and determine the mechanisms by which these interactions promote oncogenic PELP1 signaling. We identified AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, inhibited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line (J110). Furthermore, in vivo syngeneic tumor studies showed that PELP1 knockdown in J110 cells resulted in increased survival of tumor-bearing mice as compared to mice injected with control cells. These studies suggest that manipulating PELP1 location or levels has the potential to mitigate tumor progression, particularly in the context of AIB1-mediated tumorigenesis. Taken together, our data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Citation Format: Thu H. Truong, Hsiangyu Hu, Nuri A. Temiz, Kyla M. Hagen, Brian J. Girard, Nicholas J. Brady, Kathryn L. Schwertfeger, Carol A. Lange, Julie H. Ostrander. PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 532.

KLF4 functions as an oncogene in promoting cancer stem cell-like characteristics in osteosarcoma cells.

Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment, survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis. Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma. The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence, metastasis, and drug resistance. Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment. In this study, we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness. KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs: increased sphere-forming potential, enhanced levels of stemness-associated genes, great chemoresistance to adriamycin and CDDP, as well as more metastasis potential. Inversely, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis. Furthermore, KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness. Altogether, our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4-p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.

Tex10 is upregulated and promotes cancer stem cell properties and chemoresistance in hepatocellular carcinoma

ABSTRACTTestis expressed 10 (Tex10), a new core component of the pluripotency circuitry, has been reported to positively regulate embryonic stem cell (ESC) super-enhancers to promote ESC self-renewal; however, the expression and function of Tex10 in hepatocellular carcinoma (HCC) and liver cancer stem cells remains unclear. The present study was designed to investigate the expression patterns of Tex10 with immunohistochemistry, western blotting and RT-qPCR in samples from HCC patients and HCC cell lines. The results obtained show that Tex10 was highly expressed in HCC tissues, and elevated Tex10 protein levels positively correlate with the poorly differentiated carcinoma. Likewise, we found that Tex10 expression in the high-metastasis HCCLM3 potential cell line was higher than that in the low-metastasis HepG2 potential cell line, and Tex10 expression in liver cancer stem cells was also higher than that in adhered HCC cells. In addition, Tex10 knockdown decreased stem cell marker expression and drug resistance. Tex10 promoted cancer stemness through activation of the STAT3 signaling pathway. Taken together, our study demonstrates that Tex10 plays a potent carcinogenic role in HCC tumorigenesis by maintaining cancer stem cell properties through activation of the STAT3 signaling pathway and promoting chemo-resistance. Thus, targeting Tex10 may provide a novel and effective therapeutic strategy to suppress the tumorigenicity of advanced HCC.

RETRACTED ARTICLE: Platelet-derived growth factor receptor-α and -β promote cancer stem cell phenotypes in sarcomas

Sarcomas are malignant tumors derived from mesenchymal tissues and may harbor a subset of cells with cancer stem-like cell (CSC) properties. Platelet-derived growth factor receptors α and β (PDGFR-α/β) play an important role in the maintenance of mesenchymal stem cells. Here we examine the role of PDGFR-α/β in sarcoma CSCs. PDGFR-α/β activity and the effects of PDGFR-α/β inhibition were examined in 3 human sarcoma cell lines using in vitro assays and mouse xenograft models. In all three cell lines, PDGFR-α/β activity was significantly higher in cells grown as spheroids (to enrich for CSCs) and in cells sorted for CD133 expression (a marker of sarcoma CSCs). Self-renewal transcription factors Nanog, Oct4, and Slug and epithelial-to-mesenchymal transition (EMT) proteins Snail, Slug, and Zeb1 were also significantly higher in spheroids cells and CD133(+) cells. Spheroid cells and CD133(+) cells demonstrated 2.9- to 4.2-fold greater migration and invasion and resistance to doxorubicin chemotherapy. Inhibition of PDGFR-α/β in CSCs using shRNA or pharmacologic inhibitors reduced expression of certain self-renewal and EMT proteins, reduced spheroid formation by 74–82%, reduced migration and invasion by 73–80%, and reversed chemotherapy resistance. In mouse xenograft models, combining PDGFR-α/β inhibition (using shRNA or imatinib) with doxorubicin had a more-than-additive effect in blocking tumor growth, with enhanced apoptosis, especially in CD133(+) cells. These results indicate that PDGFR-α/β activity is upregulated in sarcoma CSCs and promote CSC phenotypes including migration, invasion, and chemotherapy resistance. Thus, the PDGFR-α/β pathway represents a new potential therapeutic target to reduce metastatic potential and increase chemosensitivity.

Senescence-associated reprogramming promotes cancer stemness

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Sa1199 - Acidic Microenvironment Upregulates Nanog and Oct4 Expressions Through Trpv4/Calcium Axis to Promote Cancer Stem Cell-Like Properties in Human Gastric Cancer

Sa1199 - Acidic Microenvironment Upregulates Nanog and Oct4 Expressions Through Trpv4/Calcium Axis to Promote Cancer Stem Cell-Like Properties in Human Gastric Cancer

Leptin Signaling Mediates Obesity-Associated CSC Enrichment and EMT in Preclinical TNBC Models.

Obesity is associated with poor prognosis in triple-negative breast cancer (TNBC). Preclinical models of TNBC were used to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like, triple-negative mammary tumors, received either a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat) for up to 42 weeks (n = 15/group). Mice were monitored for tumor development and euthanized when tumor diameter reached 1.5 cm. Tumoral gene expression was assessed via RNA sequencing (RNA-seq). DIO mice had greater body weight and percent body fat at termination than controls. DIO mice, versus controls, demonstrated reduced survival, increased systemic metabolic and inflammatory perturbations, upregulated tumoral CSC/EMT gene signature, elevated tumoral aldehyde dehydrogenase activity (a CSC marker), and greater leptin signaling. In cell culture experiments using TNBC cells (murine: E-Wnt and M-Wnt; human: MDA-MB-231), leptin enhanced mammosphere formation, and media supplemented with serum from DIO versus control mice increased cell viability, migration, invasion, and CSC- and EMT-related gene expression, including Foxc2, Twist2, Vim, Akt3, and Sox2 In E-Wnt cells, knockdown of leptin receptor ablated these procancer effects induced by DIO mouse serum. These findings indicate that increased leptin signaling is causally linked to obesity-associated TNBC development by promoting CSC enrichment and EMT.Implications: Leptin-associated signals impacting CSC and EMT may provide new targets and intervention strategies for decreasing TNBC burden in obese women. Mol Cancer Res; 16(5); 869-79. ©2018 AACR.

Highlights of This Issue

The present study characterizes the prevalence of microsatellite instability (MSI), tumor mutation load (TML), and PD-L1 in a large clinical cohort of gastrointestinal tumors. The clinicopathological relationship between these three features offers a more comprehensive understanding of these immune biomarkers, which may enable better patient selection and a more informed therapeutic choice to improve clinical outcome with cancer immunotherapies. These data suggest that TML-high and MSI-high (MSI-H) rates varied widely among gastrointestinal cancers. Although, MSI-H is conceivably the main driver for TML-high tumors other factors (e.g., HPV infection) may be involved.Activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR) has been implicated in various human cancers; however, mechanistic understanding of this activation remains elusive. In this Rapid Impact, by Yang and colleagues, a genome-wide, loss-of-function, luciferase reporter-based siRNA screen was performed to identify genes involved in IRE1α-XBP1 signaling regulation. Pathway analysis uncovered a subset of genes implicated in the pathogenesis of breast cancer. Several genes including BCL10, GCLM, and IGF1R correlated with worse relapse-free survival (RFS) for patients with triple-negative breast cancer (TNBC). The novel genes identified as important for XBP1 activation suggest new therapeutic paradigms for cancers in which the UPR is dysregulated.SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are frequently altered in human malignancies, the evidence showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity is limiting. Rac GTPases play a crucial role in leukemia cell engraftment, however the mechanism of Rac activation in acute myeloid leukemia (AML) is incompletely understood. Here, loss of SMARCB1 in AML associates with SWI/SNFΔ nucleation, which promotes Rac GTPase guanine nucleotide exchange factors expression, Rac activation, migration, and survival of AML cells. Thus, highlighting SWI/SNFΔ downstream signaling as an important molecular regulator in AML.Obesity is associated with poor prognosis in women with triple-negative breast cancer (TNBC). Bowers and colleagues now demonstrate that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and epithelial-to-mesenchymal transition (EMT). In a transgenic mouse model of TNBC, obesity reduced tumor-free survival and increased CSC/EMT gene expression, aldehyde dehydrogenase activity, and leptin signaling. Leptin receptor knockdown attenuated the obesity-induced CSC/EMT phenotype and Foxc2, Twist2, Vim, Akt3, and Sox2 expression in TNBC cells. Greater understanding of the leptin-related signals regulating CSC and EMT may reveal new strategies for decreasing TNBC burden in obese women.

Lnc RNA H19 is associated with poor prognosis in breast cancer patients and promotes cancer stemness.

Aldehyde dehydrogenase1 (ALDH1) is widely accepted as a stem cell marker for normal breast as well as in breast cancer. Although the clinical impact of ALDH1 was observed in our previous study, we do not know how ALDH1 affects stem cell features resulting in worsening of prognosis in breast cancer. The purpose of this study is to explore ALDH1-related gene and its function on cancer stem cell (CSC). In five cases of ALDH1-positive triple-negative breast cancer, mRNA expression profile was compared between ALDH1-positive and ALDH1-negative cells by Affymetrix microarray analysis after microdissection. Among the genes modulated in ALDH1-positive cells, we focused on H19, which encodes a long non-coding RNA, in this study. An in-vitro study was conducted with H19 siRNA in HCC1934 and iCSCL10A cell lines. The association of H19 with prognosis was examined in 180 breast cancer cases. Network analysis revealed the existence of five genes related with H19, including miR-103, miR-107, let-7, miR-29b-1, and Trx. In-vitro analysis showed that suppression of H19 using siRNA reduces sphere formation capacity in both HCC1934 and iCSCL10A cell lines. In clinical studies, H19 expression was associated with hormone negativity, tumor size, and nodal status. Patients with H19 expression had significantly poor disease-free survival (DFS) (26.3 vs. 64.8% at 5years, p = 0.001) and overall survival (OS) (28.9 vs. 68.3% at 5years, p = 0.004). The effect of H19 expression on prognosis was the most significant in triple-negative breast cancer compared to that in other subtypes (20.0 vs. 65.4% at 5years DFS, p = 0.012, 20.0 vs. 69.2% at 5years OS, p = 0.016). This study indicated that H19 was associated with stem cell phenotype in ALDH1-positive breast cancer. H19 regulates CSC and is associated with poor prognosis in breast cancer patients, particularly in triple-negative subtype.

RAB39A: a Rab small GTPase with a prominent role in cancer stemness.

RAB39A is a Rab small GTPase that localizes at distinct subcellular compartments and regulates intracellular membrane trafficking pathways in vertebrate cells. RAB39A interacts with various molecules and modulates vesicular trafficking that regulates multiple biological pathways such as neuronal differentiation and/or autophagy. Among these pathways are Hippo and Notch signallings, microtubular organization and mitophagy/autophagy. Although RAB39A has never been studied in cancer biology, it has been recently shown to promote cancer stemness and tumorigenesis. Molecular pathways regulated by RAB39A are transcriptionally maintained by the formation of molecular complex with RXRB, NCOR and HDAC that also contribute to cancer stemness. In this review, we provide current knowledge on the oncogenic function of RAB39A and summarize the effect of different microenvironments on RAB39A activity and subcellular localization in cancer cells.

Senescence Elicits Stemness: A Surprising Mechanism for Cancer Relapse

Cellular senescence is traditionally viewed as a permanent form of cell cycle arrest that restrains tumorigenesis. In a recent study in Nature, however, Milanovic etal. (2018) challenge this conventional view, showing that senescence can counterintuitively promote cancer stemness and tumor aggressiveness. This finding suggests that attacking senescence can be exploited in cancer therapy.

IGF2 induces CD133 expression in esophageal cancer cells to promote cancer stemness

Failure to eradicate cancer stem cells (CSC) during primary therapy may lead to cancer recurrence. We recently reported that CD133 is a functional biomarker for CSCs in esophageal squamous cell carcinoma (ESCC) but the molecular pathways critical for maintenance of CD133-positive CSCs are largely unknown. Here, we revealed that knockdown of IGF2 or treatment with PI3K/AKT inhibitors markedly inhibited the abilities of CD133-positive ESCC cells to self-renew, resist chemotherapeutic drugs, and form tumors. Further functional analysis identified miR-377 as a downstream regulator of PI3K/AKT signaling, and a mediator of the effects of IGF2 on CD133 expression and CSC properties. We found that the expression levels of IGF2 and CD133 were positively correlated with each other in primary ESCC, and that concurrent elevation of IGF2 and CD133 expression was significantly associated with poor patient survival. Furthermore, in vivo experiments demonstrated that IGF2-neutralizing antibody enhanced the sensitivity of tumor xenografts in nude mice to 5-fluorouracil treatment. This study underpins the importance of the IGF2-PI3K/AKT-miR-377-CD133 signaling axis in the maintenance of cancer stemness and in the development of novel therapeutic strategy for treatment of esophageal cancer.

Multi-omics analysis identifies pathways and genes involved in diffuse-type gastric carcinogenesis induced by E-cadherin, p53, and Smad4 loss in mice.

The molecular mechanisms underlying the pathogenesis of diffuse-type gastric cancer (DGC) have not been adequately explored due to a scarcity of appropriate animal models. A recently developed tool well suited for this line of investigation is the Pdx-1-Cre;Cdh1F/+ ;Trp53F/F ;Smad4F/F (pChe PS) mouse model that spontaneously develops metastatic DGC showing nearly complete E-cadherin loss. Here, we performed a proteogenomic analysis to uncover the molecular changes induced by the concurrent targeting of E-cadherin, p53, and Smad4 loss. The gene expression profiles of mouse DGCs and in vivo gastric phenotypes from various combinations of gene knockout demonstrated that these mutations collaborate to activate cancer-associated pathways to generate aggressive DGC. Of note, WNT-mediated epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM)-cytokine receptor interactions were prominently featured. In particular, the WNT target gene osteopontin (OPN) that functions as an ECM cytokine is highly upregulated. In validation experiments, OPN contributed to DGC stemness by promoting cancer stem cell (CSC) survival and chemoresistance. It was further found that Bcl-xL acts as a targetable downstream effector of OPN in DGC CSC survival. In addition, Zeb2 and thymosin-β4 (Tβ4) were identified as prime candidates as suppressors of E-cadherin expression from the remaining Cdh1 allele during DGC development. Specifically, Tβ4 suppressed E-cadherin expression and anoikis while promoting cancer cell growth and migration. Collectively, these proteogenomic analyses broaden and deepen our understanding of the contribution of key driver mutations in the stepwise carcinogenesis of DGC through novel effectors, namely OPN and Tβ4.

Epigenetic restriction of Hippo signaling by MORC2 underlies stemness of hepatocellular carcinoma cells.

The evolutionarily conserved Hippo signaling pathway is a key regulator of stem cell self-renewal, differentiation, and organ size. While alterations in Hippo signaling are causally linked to uncontrolled cell growth and a broad range of malignancies, genetic mutations in the Hippo pathway are uncommon and it is unclear how the tumor suppressor function of the Hippo pathway is disrupted in human cancers. Here, we report a novel epigenetic mechanism of Hippo inactivation in the context of hepatocellular carcinoma (HCC). We identify a member of the microrchidia (MORC) protein family, MORC2, as an inhibitor of the Hippo pathway by controlling upstream Hippo regulators, neurofibromatosis 2 (NF2) and kidney and brain protein (KIBRA). Mechanistically, MORC2 forms a complex with DNA methyltransferase 3A (DNMT3A) at the promoters of NF2 and KIBRA, leading to their DNA hyper-methylation and transcriptional repression. As a result, NF2 and KIBRA are crucial targets of MORC2 to regulate confluence-induced activation of Hippo signaling and contact inhibition of cell growth under both physiological and pathological conditions. The MORC2-NF2/KIBRA axis is critical for maintaining self-renewal, sorafenib resistance, and oncogenicity of HCC cells in vitro and in nude mice. Furthermore, MORC2 expression is elevated in HCC tissues, associated with stem-like properties of cancer cells, and disease progression in patients. Collectively, MORC2 promotes cancer stemness and tumorigenesis by facilitating DNA methylation-dependent silencing of Hippo signaling and could be a potential molecular target for cancer therapeutics.