Stress-Inducible SCAND Factors Suppress the Stress Response and Are Biomarkers for Enhanced Prognosis in Cancers.

The cell stress response is an essential system present in every cell for responding and adapting to environmental stimulations. A major program for stress response is the heat shock factor (HSF)-heat shock protein (HSP) system that maintains proteostasis in cells and promotes cancer progression. However, less is known about how the cell stress response is regulated by alternative transcription factors. Here, we show that the SCAN domain (SCAND)-containing transcription factors (SCAN-TFs) are involved in repressing the stress response in cancer. SCAND1 and SCAND2 are SCAND-only proteins that can hetero-oligomerize with SCAN-zinc finger transcription factors, such as MZF1(ZSCAN6), for accessing DNA and transcriptionally co-repressing target genes. We found that heat stress induced the expression of SCAND1, SCAND2, and MZF1 bound to HSP90 gene promoter regions in prostate cancer cells. Moreover, heat stress switched the transcript variants' expression from long noncoding RNA (lncRNA-SCAND2P) to protein-coding mRNA of SCAND2, potentially by regulating alternative splicing. High expression of HSP90AA1 correlated with poorer prognoses in several cancer types, although SCAND1 and MZF1 blocked the heat shock responsiveness of HSP90AA1 in prostate cancer cells. Consistent with this, gene expression of SCAND2, SCAND1, and MZF1 was negatively correlated with HSP90 gene expression in prostate adenocarcinoma. By searching databases of patient-derived tumor samples, we found that MZF1 and SCAND2 RNA were more highly expressed in normal tissues than in tumor tissues in several cancer types. Of note, high RNA expression of SCAND2, SCAND1, and MZF1 correlated with enhanced prognoses of pancreatic cancer and head and neck cancers. Additionally, high expression of SCAND2 RNA was correlated with better prognoses of lung adenocarcinoma and sarcoma. These data suggest that the stress-inducible SCAN-TFs can function as a feedback system, suppressing excessive stress response and inhibiting cancers.

Extracellular Vesicles: New Classification and Tumor Immunosuppression.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles carrying various types of molecules. These EV cargoes are often used as pathophysiological biomarkers and delivered to recipient cells whose fates are often altered in local and distant tissues. Classical EVs are exosomes, microvesicles, and apoptotic bodies, while recent studies discovered autophagic EVs, stressed EVs, and matrix vesicles. Here, we classify classical and new EVs and non-EV nanoparticles. We also review EVs-mediated intercellular communication between cancer cells and various types of tumor-associated cells, such as cancer-associated fibroblasts, adipocytes, blood vessels, lymphatic vessels, and immune cells. Of note, cancer EVs play crucial roles in immunosuppression, immune evasion, and immunotherapy resistance. Thus, cancer EVs change hot tumors into cold ones. Moreover, cancer EVs affect nonimmune cells to promote cellular transformation, including epithelial-to-mesenchymal transition (EMT), chemoresistance, tumor matrix production, destruction of biological barriers, angiogenesis, lymphangiogenesis, and metastatic niche formation.

SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration.

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many carcinomas depend on EMT activation, partial EMT, or hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGFß signal and EMT-inducing transcription factors, such as ZEB1/2, in tumor cells. However, reverse EMT factors are less studied. We demonstrate that prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1γ. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and ß-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway.

Optimization of production and characterization of a recombinant soluble human Cripto-1 protein inhibiting self-renewal of cancer stem cells.

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Diphenyleneiodonium efficiently inhibits the characteristics of a cancer stem cell model derived from induced pluripotent stem cells.

Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.

Cancer extracellular vesicles, tumoroid models, and tumor microenvironment.

Cancer extracellular vesicles (EVs), or exosomes, promote tumor progression through enhancing tumor growth, initiating epithelial-to-mesenchymal transition, remodeling the tumor microenvironment, and preparing metastatic niches. Three-dimensionally (3D) cultured tumoroids / spheroids aim to reproduce some aspects of tumor behavior in vitro and show increased cancer stem cell properties. These properties are transferred to their EVs that promote tumor growth. Moreover, recent tumoroid models can be furnished with aspects of the tumor microenvironment, such as vasculature, hypoxia, and extracellular matrix. This review summarizes tumor tissue culture and engineering platforms compatible with EV research. For example, the combination experiments of 3D-tumoroids and EVs have revealed multifunctional proteins loaded in EVs, such as metalloproteinases and heat shock proteins. EVs or exosomes are able to transfer their cargo molecules to recipient cells, whose fates are often largely altered. In addition, the review summarizes approaches to EV labeling technology using fluorescence and luciferase, useful for studies on EV-mediated intercellular communication, biodistribution, and metastatic niche formation.

Functional and Molecular Characters of Cancer Stem Cells Through Development to Establishment.

Cancer stem cells (CSCs) are small subpopulation sharing similar properties like normal stem such as self-renewal and differentiation potential to direct tumor growth. Last few years, scientists considered CSCs as the cause of phenotypic heterogeneity in diverse cancer types. Also, CSCs contribute to cancer metastasis and recurrence. The cellular and molecular regulators influence on the CSCs' phenotype changing their behaviors in different stages of cancer progression. CSC markers play significance roles in cancer diagnosis and characterization. We delineate the cross-talks between CSCs and the tumor microenvironment that supports their intrinsic properties including survival, stemness, quiescence and their cellular and molecular adaptation. An insight into the markers of CSCs specific to organs is described.

Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2.

Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.

MEK1/2 is a bottleneck that induces cancer stem cells to activate the PI3K/AKT pathway.

Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.

Chronic exposure to FGF2 converts iPSCs into cancer stem cells with an enhanced integrin/focal adhesion/PI3K/AKT axis.

We previously demonstrated the conversion of normal stem cells, including induced pluripotent stem cells (iPSCs), into cancer stem cells (CSCs) without genetic manipulation. Herein, we designed a meta-analysis to assess gene expression profiles in different breast cancer cell lines focusing on the secretory factors responsible for conversion. As a result, fibroblast growth factor 2 (FGF2) was found to be the best candidate in T47D and BT549 cells, of which conditioned medium was previously successful in inducing CSCs. When treated with 3.1 µg/ml FGF2, mouse iPSCs not only maintained survival without LIF for three weeks but also acquired growth ability independent of FGF2. The resultant cells exhibited expression of stemness and cancer stem cell markers, sphere-forming ability, differentiation, and tumorigenicity with malignancy. The primary cultures of the tumor confirmed the signatures of CSCs with two different phenotypes with or without GFP expression under control of the Nanog promoter. Bioinformatic analysis of gene expression profiles suggested constitutive autocrine activation of the FGF receptor, integrins, focal adhesions, and PI3K/AKT pathways. FGF2 could potently initiate cancer as a component of the inflammatory microenvironment.

Microenvironment of mammary fat pads affected the characteristics of the tumors derived from the induced cancer stem cells.

Breast cancer is the first common cause of cancer-related death in women worldwide. Since the malignancy and aggressiveness of breast cancer have been correlated with the presence of breast cancer stem cells, the establishment of a disease model with cancer stem cells is required for the development of a novel therapeutic strategy. Here, we aimed to evaluate the availability of cancer stem cell models developed from mouse induced pluripotent stem cells with the conditioned medium of different subtypes of breast cancer cell lines, the hormonal-responsive T47D cell line and the triple-negative breast cancer BT549 cell line, to generate in vivo tumor models. When transplanted into the mammary fat pads of BALB/c nude mice, these two model cells formed malignant tumors exhibiting pronounced histopathological characteristics similar to breast cancers. Serial transplantation of the primary cultured cells into mammary fat pads evoked the same features of breast cancer, while this result was perturbed following subcutaneous transplantation. The tumors formed in the mammary fat pads exhibited immune reactivities to prolactin receptor, progesterone receptor, green florescent protein, Ki67, CD44, estrogen receptor α/ß and cytokeratin 8, while all of the tumors and their derived primary cells exhibited immunoreactivity to estrogen receptor α/ß and cytokeratin 8. Cancer stem cells can be developed from pluripotent stem cells via the secretory factors of cancer-derived cells with the capacity to inherit tissue specificity. However, cancer stem cells should be plastic enough to be affected by the microenvironment of specific tissues. In summary, we successfully established a breast cancer tumor model using mouse induced pluripotent stem cells developed from normal fibroblasts without genetic manipulation.

Syndecan-1 (CD138) as a Pathogenesis Factor and Therapeutic Target in Breast Cancer.

The successive stages of breast cancer growth and dissemination depend on cell-autonomous factors and the communication between tumor cells and their surrounding cellular and extracellular matrix microenvironment. The cell surface heparan sulfate proteoglycan Syndecan-1 is dysregulated both in tumor cells and cells of the breast tumor stroma, indicating a potential role in the pathogenesis of this most frequent malignancy in women. Indeed, Syndecan-1 interacts with numerous ligands and receptors relevant to tumor progression, affecting processes as diverse as cancer stem cell function, cell proliferation, apoptosis, cell adhesion, migration and invasion, tumor angiogenesis, and leukocyte function in the tumor stroma. The present review summarizes the current understanding of breast carcinogenesis in correlation with their Syndecan-1 expression, involved mechanisms, and proposed therapeutic strategies against Syndecan-1-related malignancy.