Enhanced Stem Cell Properties Research Articles

A microprotein N1DARP encoded by LINC00261 promotes Notch1 intracellular domain (N1ICD) degradation via disrupting USP10-N1ICD interaction to inhibit chemoresistance in Notch1-hyperactivated pancreatic cancer

The extensively activated Notch signaling pathway in pancreatic cancer cells is important in carcinogenesis, chemoresistance, and recurrence. Targeting this pathway is a promising therapeutic strategy for pancreatic cancer; however, few successful approaches have been reported, and currently used molecular inhibitors of this pathway exhibit limited clinical benefits. In this study, we identified a previously uncharacterized microprotein, Notch1 degradation-associated regulatory polypeptide (N1DARP), encoded by LINC00261. N1DARP knockout accelerated tumor progression and enhanced stem cell properties in pancreatic cancer organoids and LSL-Kras, LSL-Trp53, and Pdx1-Cre (KPC) mice. Mechanistically, N1DARP suppressed canonical and non-canonical Notch1 pathways by competitively disrupting the interaction between N1ICD and ubiquitin-specific peptidase 10 (USP10), thereby promoting K11- and K48-linked polyubiquitination of N1ICD. To evaluate the therapeutic potential of N1DARP, we designed a cell-penetrating stapled peptide, SAH-mAH2-5, with a helical structure similar to that of N1DARP that confers remarkable physicochemical stability. SAH-mAH2-5 interacted with and promoted the proteasome-mediated degradation of N1ICD. SAH-mAH2-5 injection provided substantial therapeutic benefits with limited off-target and systemic adverse effects in Notch1-activated pancreatic cancer models. Taken together, these findings confirm that N1DARP acts as a tumor suppressor and chemosensitizer by regulating USP10-Notch1 oncogenic signaling, and suggest a promising therapeutic strategy targeting the N1DARP–N1ICD interaction in Notch1-activated pancreatic cancer.

The Emerging Landscapes of Long Noncoding RNA in Thyroid Carcinoma: Biological Functions and Clinical Significance.

Thyroid carcinoma (TC) is one of the most prevalent primary endocrine tumors, and its incidence is steadily and gradually increasing worldwide. Accumulating evidence has revealed the critical functions of long noncoding RNAs (lncRNAs) in the tumorigenesis and development of TC. Many TC-associated lncRNAs have been documented to be implicated in TC malignant behaviors, including abnormal cell proliferation, enhanced stem cell properties and aggressiveness, and resistance to therapeutics, through interaction with proteins, DNA, or RNA or encoding small peptides. Therefore, further elucidating the lncRNA dysregulation sheds additional insights into TC tumorigenesis and progression and opens new avenues for the early diagnosis and clinical therapy of TC. In this review, we summarize the abnormal expression of lncRNA in TC and the fundamental characteristics in TC tumorigenesis and development. Additionally, we introduce the potential prognostic and therapeutic significance of lncRNAs in TC.

Axin1 translocates into the nucleus in hepatocyte growth factor induced epithelial-mesenchymal transition

Axin1 is a scaffold protein belonging to a destruction complex that degrades β-catenin. As an inhibitor of Wnt signaling, Axin1 is one of the most widely studied therapeutic targets in cancer treatment, particularly as targets for Tankyrase inhibitors. Axin1 has long been considered to be uninvolved in transforming growth factor beta induced epithelial-mesenchymal transition (EMT); the latter is known to transform cancer cells to a more aggressive phenotype that show increased cell mobility, drug-resistance and enhanced stem cell properties by remaining in the cytoplasm.In this study, the effects of overexpression of Axin1 on hepatocyte growth factor (HGF) induced EMT in HepG2 cells was investigated through analyses of in-cell western, morphological changes, cell growth curve, population doubling time and immunofluorescence staining.It was observed that Axin1 overexpression in HepG2 cells inhibited cellular growth. HGF treatment of Axin1 overexpressing cells resulted in the induction of EMT with the cells acquiring a fibroblast-like spindle morphology. This is the first report to describe that Axin1 translocated to the nucleus during HGF induced EMT, which is how this protein may exert EMT promoting functions. The result of this observation may rise the questions on the safety of Tankyrase inhibitors which stabilize Axin1 for cancer therapy.

Identification of HSC/MPP expansion units in fetal liver by single-cell spatiotemporal transcriptomics.

Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP 'pocket-like' units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage-HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell-cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

Context-dependent EMT programs in cancer metastasis.

Epithelial-mesenchymal transition (EMT) is a developmental process whereby stationary, adherent cells acquire the ability to migrate. EMT is critical for dramatic cellular movements during embryogenesis; however, tumor cells can reactivate EMT programs, which increases their aggressiveness. In addition to motility, EMT is associated with enhanced stem cell properties and drug resistance; thus it can drive metastasis, tumor recurrence, and therapy resistance in the context of cancer. However, the precise requirements for EMT in metastasis have not been fully delineated, with different tumor types relying on discrete EMT effectors. Most tumor cells do not undergo a full EMT, but rather adopt some qualities of mesenchymal cells and maintain some epithelial characteristics. Emerging evidence suggests that partial EMT can drive distinct migratory properties and enhance the epithelial-mesenchymal plasticity of cancer cells as well as cell fate plasticity. This review discusses the diverse regulatory mechanisms and functional consequences of EMT, with an emphasis on the importance of partial EMT.

MiR-378 promoted cell proliferation and inhibited apoptosis by enhanced stem cell properties in chronic myeloid leukemia K562 cells

Dysregulation of miR-378 has been found in diverse types of tumors as well as in leukemia. The role of miR-378 in chronic myeloid leukemia (CML) remains unclear. The aim of the study was to reveal the potential effects of miR-378 in the pathological process and progress in CML. Our results showed general level of miR-378 was significant higher in CML patients compared to controls. Overexpression of miR-378 dramatically promoted cell proliferation and drug-resistance. Additionally, apoptosis was inhibited in cells transfected with miR-378. More and bigger stem cell sphere formation was observed in miR-378 transfected cells. Furthermore, enhanced expression of miR-378 was associated with upregulation of stem-cell makers OCT4 and c-Myc. Further study validated that miR-378 inhibited the expression of FUS1. Our research demonstrated the oncogenic nature of miR-378 in CML, and might contribute to the progress of CML.

Identification of TIM-3/Gal-9 Autocrine Loop As a Novel Wnt-Ligands Independent Machinery for the Constitutive Activation of Canonical Wnt Pathway in AML-LSCs

We originally identified T-cell immunoglobulin mucin-3 (TIM-3) as a leukemic stem cells (LSCs)-specific surface molecule and a useful marker for discriminating LSCs from hematopoietic stem cells (HSCs). Furthermore, we recently identified an unique autocrine loop composed of TIM-3 and its ligand galectin-9 (Gal-9). This TIM-3/Gal-9 autocrine loop enhances self-renewal capacity of AML-LSCs and contributes to leukemia progression(Kikushige et al., Cell Stem Cell 2015).To clarify the molecular mechanism how TIM-3 signaling enhances stem cell properties of AML-LSCs, we performed shRNA-mediated knock-down(KD) of TIM-3 in KASUMI-3 (TIM-3+ AML cell line). As a result, TIM-3-KD significantly reduce the proliferation of KASUMI-3. To investigate how TIM-3-KD attenuated leukemia propagation, we compared gene expression profile of scramble-infected control and TIM-3-KD KASUMI-3 cells. Gene Set Enrichment Analysis (GSEA) revealed TIM-3-KD resulted in the significaltly attenuated expression of HSCs and LSCs-related genes (Eppert et al., Nature Med 2011) and canonical Wnt pathway-related genes (BioCarta). Since canonical Wnt pathway is known to regulate stem cell properties via inducing the nucleus accumulation ofβ-catenin in many types of stem cells including HSCs, we next examined the β-catenin level in TIM-3-KD-KASUMI-3 cells. Array scan analysis revealed that KD of TIM-3 significantly impaired the the nucleus accumulation of β-catenin in KASUMI-3 cells. In various types of cancers, the constitutive activation of β-catenin is one of the most common oncogenic signatures, and it is mainly driven by somatic mutations in the genes related to the canonical Wnt pathway. However, such mutations are rarely detected in AML.We, therefore, hypothesized that TIM-3/Gal-9 autocrine loop signaling might be a novel mutation-independent machinery for the constitutive activation of canonical Wnt pathway in primary AML.To test this hypothethis, we first evaluated the phosphorylation status of LDL receptor-related protein 6 (LRP6), because LRP6 phosphorylation is a crucial step for canonical Wnt pathway activation. Surprisingly, Gal-9 stimulation induced phosphorylation of LRP6 and subsequent β-catenin accumulation in primary TIM-3+ AML cells even in the absence of Wnt ligand. On the other hand, LRP6 phosphorylation induced by Gal-9 stimulation was canceled by the blocking antibody to TIM-3 (F38-2E2). These results indicate that Gal-9 stimulation should induce canonical Wnt pathway activation via TIM-3 dependent manner in primary AML cells. Since Gal-9 ligation to TIM-3 is known to induce phosphorylation of tyrosine residues of TIM-3 cytoplasmic tail, leading to the recruitment and activation of several Src family kinases (SFKs) for downstream signal transduction. We found that TIM-3/Gal-9 interaction recruited hematopoietic cell kinase (HCK), a member of SFKs, to TIM-3 and activated HCK by phosphorylation of the SH1 domain (Y410 in HCK) in TIM-3+ AML cells. Furthermore, we confirmed a specific HCK inhibitor A-419259 efficiently blocked the LRP6 phosphorylation and subsequent β-catenin accumulation induced by TIM-3/Gal-9 interaction in primary AML cells. These results suggest that the canonical Wnt pathway activation induced by Gal-9 ligation to TIM-3 is mainly mediated by HCK in primary AML cells.We next focused on p120-catenin as a bridging molecule between HCK activation and LRP6 phosphorylation because, as previously reported, this molecule can be directly activated by SFKs and the activation of this molecule is an initial step for canonical Wnt pathway activation preceding LRP6 phosphorylation. We found that Gal-9 ligation to TIM-3 immediately induced the direct association of HCK and p120-catenin, leading to the activation of p120-catenin in TIM-3+ AML cells. We also confirmed that specific inhibition of HCK completely abrogated the activation of p120-catenin and subsequent canonical Wnt pathway activation in primary TIM-3+ AML cells, indicating that the activation of p120-catenin mediated by HCK is a bridging moleculer machinery for connecting TIM-3 signaling and previously identified canonical Wnt pathway. Since TIM-3 as well as HCK is specifically expressed in LSCs but not in HSCs, we conclude that TIM-3/Gal-9 autocrine loop is a novel Wnt-ligands independent machinery for the constitutive activation of canonical Wnt pathway specific for AML-LSCs. (summarized in figure) [Display omitted] DisclosuresAkashi:Celgene: Research Funding, Speakers Bureau; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Taiho Pharmaceutical: Research Funding; MSD: Research Funding; Asahi-kasei: Research Funding; Chugai Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis pharma: Research Funding; Eisai: Research Funding; sanofi: Research Funding; Astellas Pharma: Research Funding.

Dedifferentiation process driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer.

Cancer stem cells (CSCs) possess the capacity for self-renewal and the potential to differentiate into non-CSCs. The recent discoveries of dynamic equilibrium between CSCs and non-CSCs revealed the significance of acquiring CSC-like properties in non-CSCs as an important process in progression of cancer. The mechanism underlying acquisition of CSC-like properties has mainly been investigated in the context of epithelial-mesenchymal transition. Here, we demonstrate the dedifferentiation process may be an alternative mechanism in acquisition of CSC-like properties in human colorectal cancer cells. By exploring the single-cell gene expression analysis of organoids developed from CD44+ CSCs, we identified TWIST1 as a key molecule for maintaining the undifferentiated state of cancer cells. Consistent with the finding, we found that TGF-beta signaling pathway, a regulator of TWIST1, was specifically activated in the undifferentiated CD44+ CSCs in human colorectal cancer using microarray-based gene expression analysis and quantitative pathology imaging system. Furthermore, we showed that external stimulation with TGF-beta and the induction of TWIST1 converted CD44- non-CSCs into the undifferentiated CD44+ CSCs, leading to the significant increment of CSCs in xenograft models. This study strongly suggests dedifferentiation driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer.

Abstract LB-279: N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells

Abstract New generation of anti-androgen treatments such as enzalutamide and abiraterone are improving survival of men with advanced and metastatic prostate cancers, but resistance to these inevitably arises. The molecular mechanisms governing the emergence of treatment resistance in castration resistant prostate cancer (CRPC) patients are not well understood. Recent experience suggests that tumor regeneration from castration-resistant stem-like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem-like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial-mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. We previously showed that N-cadherin, a marker of EMT, promotes CRPC progression and that there was a correlation between N-cadherin expression and the expression of stem cell associated markers. This leads to hypothesis that N-cadherin may promote CRPC by enhancing tumorige esis/tumor initiating properties of cancer cells in a castrate environment. N-cadherin may confer tumor initiating or stem-like properties to prostate cancer cells perhaps uniquely or specific to in a castrate environment. Here, we verified that EMT is linked to stem cells in both normal prostate and CRPC. We further demonstrate that N-cadherin is involved in increased stem cell properties in normal mouse and human prostates from clinical samples in an androgen-deprived environment. Castration elevates the expression of N-cadherin and stem cell marker Trop2, accompanied with enhanced stem cell properties of luminal population in normal mouse prostates. N-cadherin upregulates stem cell properties in prostate cancer cells in the absence of androgen and CRPC tumors. N-cadherin-positive cells from CRPC tumors behave as stem cells. Overexpression of N-cadherin enhances ALDH activity in a panel of prostate cancer cell lines. Our findings reveal an important role of N-cadherin in prostate cancer and stem cells, and provide useful information of EMT-related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to next-generation AR antagonists. Citation Format: Shu Lin, Tanya Stonyanova, Evelyn Kono, Jaclyn Matsuura, Naoko Kobayashi, Joyce Yamashiro, Dai Hong, Owen N. Witte, Andrew S. Goldstein, Robert E. Reiter. N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-279.

The senescent microenvironment promotes the emergence of heterogeneous cancer stem-like cells.

There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state.

Suberoylanilide hydroxamic acid (SAHA) reverses chemoresistance in head and neck cancer cells by targeting cancer stem cells via the downregulation of nanog.

Acquisition of chemoresistance and metastatic phenotype are the major causes of treatment failure and mortality in head and neck squamous cell carcinoma (HNSCC) patients. Histone deacetylases (HDACs) have been shown to be overexpressed in many tumor types and directly linked to poor prognosis. In this study, we demonstrate that HDACs are markedly elevated in HNSCC. HDACs expression was further increase in cisplatin resistant cell lines (CisR). In addition, cisplatin-resistant cells showed enhanced stem cell properties and tumor metastasis. Depletion of HDAC1 and 2 in CisR cell lines significantly reversed cisplatin resistance and tumorsphere formation. Next, we tested the efficacy of Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, by using both in vitro and in vivo models. SAHA significantly inhibited cell proliferation and synergistically enhanced the anti-proliferative effects of cisplatin. In addition, SAHA significantly decreased tumorsphere formation by markedly reducing nanog expression. In a SCID mouse xenograft model, SAHA significantly enhanced the anti-tumor effects of cisplatin treatment with no added systemic toxicity. Furthermore, SAHA and cisplatin combination treatment significantly decreased tumor metastasis and nanog expression, in vivo. Taken together, our results suggest that targeting HDACs with SAHA could be an effective treatment strategy for the treatment of HNSCC patients.

NKX2.2 Suppresses Self-Renewal of Glioma-Initiating Cells

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16(Ink4a)/p19(Arf) led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.

Blocking cancer metastasis with TGF-beta antagonists

I of epithelial-mesechymal transition (EMT) has been shown to confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT. We found that chemotherapeutic drug doxorubicin activates TGFβ signaling in breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced stem cell properties in the murine 4T1 breast cancer cells in vitro, which were inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). Th ese observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the effi cacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was signifi cantly blocked when TβR1-KI was administered in combination with doxorubicin. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-eff ects of doxorubicin, and improve its effi cacy in the inhibition of breast cancer growth and metastasis.