Transcription Factor SALL4 Research Articles

Higher expression of SALL4-A isoform is correlated with worse outcomes and progression of the disease in subtype of testicular germ cell tumours

Background The transcription factor SALL4 is associated with embryonic pluripotency and has proposed as a novel immunohistochemistry (IHC) marker for diagnosing germ cell tumours. SALL4 comprises three isoforms, and SALL4-A being the full-length isoform. Studying its isoforms could revolutionize testicular cancer prognosis and subtype differentiation. Methods The expression and clinical significance of isoform ‘A’ of SALL4 was evaluated in 124 testicular germ cell tumours (TGCTs) subtypes, adjacent 67 normal tissues and 22 benign tumours, using immunohistochemistry on tissue microarrays (TMA). Results A statistically significant higher expression of nuclear and cytoplasmic SALL4-A was detected in TGCTs histological subtypes and benign tumours compared to the normal tissues. Seminoma and yolk sac tumours had the highest nuclear and cytoplasmic expression of SALL4-A. A significant correlation was detected between the higher nuclear expression of SALL4-A and increased pT stages (P = 0.026) in seminomas. Whereas in embryonal carcinomas, cytoplasmic expression of SALL4-A was associated with the tumour recurrence (P = 0.04) and invasion of the epididymis (P = 0.011). Conclusions SALL4-A isoform expression in the cytoplasm and nucleus of TGCTs may be associated with histological differentiation. In the seminoma subtype of TGCTs, higher expression of SALL4-A may be used as a predictive indicator of poorer outcomes and prognosis.

Establishment, characterization, and drug screening of low-passage patient individual non-small cell lung cancer in vitro models including the rare pleomorphic subentity.

For pre-clinical drug development and precision oncology research, robust cancer cell models are essential. Patient-derived models in low passages retain more genetic and phenotypic characteristics of their original tumors than conventional cancer cell lines. Subentity, individual genetics, and heterogeneity greatly influence drug sensitivity and clinical outcome. Here, we report on the establishment and characterization of three patient-derived cell lines (PDCs) of different subentities of non-small cell lung cancer (NSCLC): adeno-, squamous cell, and pleomorphic carcinoma. The in-depth characterization of our PDCs included phenotype, proliferation, surface protein expression, invasion, and migration behavior as well as whole-exome and RNA sequencing. Additionally, in vitro drug sensitivity towards standard-of-care chemotherapeutic regimens was evaluated. The pathological and molecular properties of the patients' tumors were preserved in the PDC models HROLu22, HROLu55, and HROBML01. All cell lines expressed HLA I, while none were positive for HLA II. The epithelial cell marker CD326 and the lung tumor markers CCDC59, LYPD3, and DSG3 were also detected. The most frequently mutated genes included TP53, MXRA5, MUC16, and MUC19. Among the most overexpressed genes in tumor cells compared to normal tissue were the transcription factors HOXB9, SIM2, ZIC5, SP8, TFAP2A, FOXE1, HOXB13, and SALL4; the cancer testis antigen CT83; and the cytokine IL23A. The most downregulated genes on the RNA level encode the long non-coding RNA LANCL1-AS1, LINC00670, BANCR, and LOC100652999; the regulator of angiogenesis ANGPT4; the signaling molecules PLA2G1B and RS1; and the immune modulator SFTPD. Furthermore, neither pre-existing therapy resistances nor drug antagonistic effects could be observed. In summary, we successfully established three novel NSCLC PDC models from an adeno-, a squamous cell, and a pleomorphic carcinoma. Of note, NSCLC cell models of the pleomorphic subentity are very rare. The detailed characterization including molecular, morphological, and drug-sensitivity profiling makes these models valuable pre-clinical tools for drug development applications and research on precision cancer therapy. The pleomorphic model additionally enables research on a functional and cell-based level of this rare NCSLC subentity.

Novel, thalidomide-like, non-cereblon binding drug tetrafluorobornylphthalimide mitigates inflammation and brain injury

BackgroundQuelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurological disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs.MethodsTFBP/TFNBP were synthesized and evaluated for cereblon binding and anti-inflammatory actions in human and rodent cell cultures. Teratogenic potential was assessed in chicken embryos, and in vivo anti-inflammatory actions in rodents challenged with either lipopolysaccharide (LPS) or controlled cortical impact (CCI) moderate traumatic brain injury (TBI). Molecular modeling was performed to provide insight into drug/cereblon binding interactions.ResultsTFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with LPS, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 h post-injury following CCI TBI. Compared to vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2-weeks post-injury. Behavioral evaluations at 1- and 2-weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice.ConclusionTFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurological disorders involving a neuroinflammatory component.Graphical

Role of SALL4 in HER2+ Breast Cancer Progression: Regulating PI3K/AKT Pathway.

Treatment for the HER2+ breast cancer subtype is still unsatisfactory, despite breakthroughs in research. The discovery of various new molecular mechanisms of transcription factors may help to make treatment regimens more effective. The transcription factor SALL4 has been related to aggressiveness and resistance therapy in cancer. Its molecular mechanisms and involvement in various signaling pathways are unknown in the HER2+ breast cancer subtype. In this study, we have evaluated the implication of SALL4 in the HER2+ subtype through its expression in patients' samples and gain and loss of function in HER2+ cell lines. We found higher SALL4 expression in breast cancer tissues compared to healthy tissue. Interestingly, high SALL4 expression was associated with disease relapse and poor patient survival. In HER2+ cell lines, transient overexpression of SALL4 modulates PI3K/AKT signaling through regulating PTEN expression and BCL2, which increases cell survival and proliferation while reducing the efficacy of trastuzumab. SALL4 has also been observed to regulate the epithelial-mesenchymal transition and stemness features. SALL4 overexpression significantly reduced the epithelial markers E-cadherin, while it increased the mesenchymal markers β-catenin, vimentin and fibronectin. Furthermore, it has been also observed an increased expression of MYC, an essential transcription factor for regulating epithelial-mesenchymal transition and/or cancer stem cells. Our study demonstrates, for the first time, the importance of SALL4 in the HER2+ subtype and partial regulation of trastuzumab sensitivity. It provides a viable molecular mechanism-driven therapeutic strategy for an important subset of HER2-overexpressing patients whose malignancies are mediated by SALL4 expression.

Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy.

Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells (CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important. Cancerous cell suspension isolated from patients with GC was cultured in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated. Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells. We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, especially in patients with advanced disease.

A regulatory role for CHD4 in maintenance of the spermatogonial stem cell pool.

SummaryMaintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of “self-renewal” genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool.

The Interplay between Transcription Factor SALL4 and Histone Modifiers in Hematopoietic Stem and Progenitor Cells.

The Interplay between Transcription Factor SALL4 and Histone Modifiers in Hematopoietic Stem and Progenitor Cells.

Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

SUMMARYThe zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.

Zinc Finger Protein SALL4 Functions Through an AT-Rich Motif to Regulate Gene Expression

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, down-regulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive target for designing therapeutic strategies. However, whether SALL4 binds DNA directly to regulate gene expression is unclear and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) experiments, we identified and validated the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA-seq analyses after SALL4 knockdown, we discovered hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of Histone 3 Lysine 9-specific Demethylases (KDMs). Taken together, these results elucidated the mechanism of SALL4 DNA binding and revealed novel pathways and molecules to target in SALL4-dependent tumors.

Abstract 1788: A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in cancer

Abstract Transcription factors are important drivers of cancer but the development of therapeutics against these factors has had limited success. We developed a stringent high-throughput chemical genetic screening platform to identify compounds that target oncogenic transcription factor SALL4 dependency in liver cancer. The platform comprises SALL4 low- and high-expressing endogenous and engineered isogenic liver cancer cell lines. Unexpectedly, from screening 21,575 natural product extracts, the top hits were four oxidative phosphorylation inhibitors that selectively reduced SALL4-dependent cell viability. The ATP synthase inhibitor oligomycin suppressed SALL4-expressing cancer in lung and liver cancer cell culture models, and in patient-derived xenograft models of liver cancer. Oligomycin also synergized with sorafenib, the standard-of-care targeted therapy in liver cancer, to effectively suppress SALL4-driven tumorigenesis in vivo. When aberrantly expressed in cancer, SALL4 binds ~50% of mitochondrial genes, including many oxidative phosphorylation genes, to predominantly upregulate their expression. SALL4 upregulation also alters the levels of oxidative phosphorylation-related metabolites and functionally increases oxidative phosphorylation activity. Application of our endogenous/isogenic transcription factor-screening platform revealed a therapeutically actionable oxidative phosphorylation vulnerability in SALL4-expressing cancers. Citation Format: Justin L. Tan, Feng Li, Joanna Z. Yeo, Kol Jia Yong, Mahmoud A. Bassal, Guo Hao Ng, May Yin Lee, Chung Yan Leong, Hong Kee Tan, Chan-Shuo Wu, Bee Hui Liu, Hon Man Chan, Zi Hui Tan, Yun Shen Chan, Siyu Wang, Zhi Han Lim, Tan Boon Toh, Lissa Hooi, Kia Ngee Low, Siming Ma, Nikki R. Kong, Alicia J. Stein, Yue Wu, Matan T. Thangavelu, Atsushi Suzuki, Giridharan Periyasamy, John M. Asara, Yock Young Dan, Glenn K. Bonney, Edward K. Chow, Guo-Dong Lu, Huck Hui Ng, Yoganathan Kanagasundaram, Siew Bee Ng, Wai Leong Tam, Li Chai, Daniel G. Tenen. A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1788.

Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer.

In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl2-induced hypoxia in breast cancer further promotes HIF-1α mRNA and protein expression while reducing VHL expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (COX-2, EP4, NFκB1, VEGFA) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) expression by downregulation of transcription factors NR2C2, SALL4, and ZNF207. Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, HIF-1α expression is significantly higher in human breast tumors (n = 96) compared to non-cancerous control tissues (n = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, HIF-1α expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between HIF-1α and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.

VHL mutation-mediated SALL4 overexpression promotes tumorigenesis and vascularization of clear cell renal cell carcinoma via Akt/GSK-3\u03b2 signaling

BackgroundAlthough ongoing development of therapeutic strategies contributes to the improvements in clinical management, clear cell renal cell carcinoma (ccRCC) deaths originate mainly from radiochemoresistant and metastatic disease. Transcription factor SALL4 has been implicated in tumorigenesis and metastasis of multiple cancers. However, it is not known whether SALL4 is involved in the pathogenesis of ccRCC.MethodsAnalyses of clinical specimen and publicly available datasets were performed to determine the expression level and clinical significance of SALL4 in ccRCC. The influence of SALL4 expression on ccRCC tumor growth, metastasis and vascularity was evaluated through a series of in vitro and in vivo experiments. Western blotting, immunofluorescence staining and integrative database analysis were carried out to investigate the underlying mechanism for SALL4-mediated oncogenic activities in ccRCC.ResultsSALL4 expression was increased in ccRCC and positively correlated with tumor progression and poor prognosis. SALL4 could promote ccRCC cell proliferation, colony formation, cell cycle progression, migration, invasion and tumorigenicity and inhibit cell senescence. Further investigation revealed a widespread association of SALL4 with individual gene transcription and the involvement of SALL4 in endothelium development and vasculogenesis. In the context of ccRCC, SALL4 promoted tumor vascularization by recruiting endothelial cells. In addition, we found that SALL4 could exert its tumor-promoting effect via modulating Akt/GSK-3β axis and VEGFA expression. VHL mutation and DNA hypomethylation may be involved in the upregulation of SALL4 in ccRCC.ConclusionsOverall, our results provide evidence that upregulated SALL4 can function as a crucial regulator of tumor pathogenesis and progression in ccRCC, thus offering potential therapeutic strategies for future treatment.

Crystal structure of the SALL4-pomalidomide-cereblon-DDB1 complex.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation

Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP. In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Abstract P2-01-11: A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells

Abstract Metastasis inhibition may improve survival of breast cancer patients. Our previous study revealed that a transcription factor SALL4 promotes cell migration for metastasis through up-regulation of integrin α6β1 in metastatic basal-like breast cancer cells. In these cells, integrin α6β1 modulates Rho GTPase activity, which enhances focal adhesion dynamics for cell migration. We therefore focused on integrin α6 as a therapeutic target for metastasis. We investigated the expression level of integrin α6 in breast cancer cell lines and a tissue microarray of breast cancer patients. In both experiments, we observed higher integrin α6 expression in basal-like breast cancer cells than in luminal ones. We performed shRNA-mediated integrin α6 knockdown in basal-like breast cancer cells and conducted Boyden chamber assays. In the results, reduced cell migration was observed in integrin α6 knocked-down cells. Reciprocally, we introduced integrin α6 overexpression in low-migratory luminal breast cancer cell lines and observed increased cell migration. These results indicate that integrin α6 promotes cell migration in breast cancer cells. To identify the functional residue of integrin α6, we analyzed its amino acid sequence by in silico analyses. First, we extracted integrin α6 specific residues in integrin α family in the human genome. Then, we compared integrin α6 sequences among vertebrates. In the result, we obtained an integrin α6-specific and vertebrate-conserved sequence, Asp-358, as a candidate for the functional residue. To inhibit integrin α6 function, we designed an 8-amino acid peptide with the sequence around Asp-358. Basal-like breast cancer cells treated with the peptide showed reduced cell migration in a dose-dependent manner. The peptide did not reduce cell migration in integrin α6 knocked-down cells, suggesting that the peptide inhibits integrin α6 function. To determine whether the peptide inhibits metastasis, we performed zebrafish metastasis assays, and observed reduced metastasis rate in the peptide-treated group. These results indicate that the peptide inhibits metastasis through reduction in cell migration. To understand the effect of the peptide, we performed chemical cross-liking assay in a basal-like breast cancer cell line. Protein samples from the cells treated with a cross-linker showed bands of integrin α6 complexes, and the intensities of these bands were reduced in the peptide-treated group. Moreover, we immunostained cells with an antibody for focal adhesion marker, phospho-paxillin, because integrins regulates focal adhesion formation for cell migration. In the results, the peptide reduced the number of focal adhesions. These observations indicate that the peptide inhibits integrin α6 function. In the future, our findings may contribute to development of a metastasis inhibition therapy. Citation Format: Itou J, Tanaka S, Senda N, Iida A, Sehara-Fujisawa A, Sato F, Toi M. A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-11.

A new target for thalidomide.

One mechanism of thalidomide teratogenicity involves binding to the CUL4–CRBN E3 ubiquitin ligase complex, which then mediates the degradation of transcription factors. New studies reveal that species-specific variants of the transcription factor SALL4 are differentially ubiquitinated and degraded via the thalidomide-bound complex.

PO-163 Identification of candidate genes underlying soft tissue sarcoma progression using a progression series of murine fibrosarcoma cell lines

IntroductionThe transcription factor Sall4 is a well-known developmental regulator involved in embryonic patterning and stem cell maintenance. More recently Sall4 has been suggested to be an oncofetal gene due to being expressed in many fetal tissues and malignant tumours but only rarely in normal adult tissues. The re-expression of such genes in cancer might reflect crucial processes during development, which are reactivated in neoplasias such as increased migratory and invasive capacities. Sall4 is discussed as novel marker and target in some solid tumours and haematological malignancies, however its role in melanoma is unknown.Material and methodsTo address a putative role of Sall4 in melanoma, we used the Tyr::NrasQ61KINK4a−/− murine melanoma model, which spontaneously develops metastatic melanoma. Those mice were crossed with Tyr::CreERT2Sall4lox/loxR26R::GFP mice, allowing us to conditionally ablate Sall4 in melanocytes and to trace Cre activity via GFP expression. We further used human melanoma patient-derived primary cell cultures for in vitro experiments and the same cell lines as xenographs in immunocompromised mice.Results and discussionsMice lacking Sall4 did not form primary tumours compared to the control animals however strikingly had more GFP +melanoma metastases in the lungs. Sall4lox/wt animals formed tumours like control animals but also showed more lung metastases. Through RNA Sequencing on siSALL4-treated human melanoma cell lines we found that SALL4 decrease correlates with upregulation of melanoma invasiveness genes and functionally increased invasiveness in vitro. We further found by Co-IP that SALL4 binds to members of the histone deacetylase family (HDACs) and by running a ChIP-Seq for H3K27ac, we could indeed verify that knocking down SALL4 lead to increased histone acetylation of enhancer regions of the mentioned invasiveness genes. Interestingly, HDAC inhibitor (HDACi) treatment in vitro resulted in an invasiveness gene expression pattern very similar to SALL4 knock down and HDACi treatment of xenograph tumours also lead to an expression profile hinting towards increased invasiveness in vivo.ConclusionAll in all we found that downregulation of Sall4 in melanoma leads to increased acetylation and expression of invasiveness genes in vitro and increased metastasis in vivo, with both phenotypes being closely mimicked by HDAC inhibitor application.

MiR-508 Defines the Stem-like/Mesenchymal Subtype in Colorectal Cancer.

Colorectal cancer includes an invasive stem-like/mesenchymal subtype, but its genetic drivers, functional, and clinical relevance are uncharacterized. Here we report the definition of an altered miRNA signature defining this subtype that includes a major genomic loss of miR-508. Mechanistic investigations showed that this miRNA affected the expression of cadherin CDH1 and the transcription factors ZEB1, SALL4, and BMI1. Loss of miR-508 in colorectal cancer was associated with upregulation of the novel hypoxia-induced long noncoding RNA AK000053. Ectopic expression of miR-508 in colorectal cancer cells blunted epithelial-to-mesenchymal transition (EMT), stemness, migration, and invasive capacity in vitro and in vivo In clinical colorectal cancer specimens, expression of miR-508 negatively correlated with stemness and EMT-associated gene expression and positively correlated with patient survival. Overall, our results showed that miR-508 is a key functional determinant of the stem-like/mesenchymal colorectal cancer subtype and a candidate therapeutic target for its treatment.Significance: These results define a key functional determinant of a stem-like/mesenchymal subtype of colorectal cancers and a candidate therapeutic target for its treatment. Cancer Res; 78(7); 1751-65. ©2018 AACR.

Germline Stem Cell Activity Is Sustained by SALL4-Dependent Silencing ofDistinct Tumor Suppressor Genes.

SummarySustained spermatogenesis in adult males and fertility recovery following germ cell depletion are dependent on undifferentiated spermatogonia. We previously demonstrated a key role for the transcription factor SALL4 in spermatogonial differentiation. However, whether SALL4 has broader roles within spermatogonia remains unclear despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. Through development of inducible knockout models, we show that short-term integrity of differentiating but not undifferentiated populations requires SALL4. However, SALL4 loss was associated with long-term functional decline of undifferentiated spermatogonia and disrupted stem cell-driven regeneration. Mechanistically, SALL4 associated with the NuRD co-repressor and repressed expression of the tumor suppressor genes Foxl1 and Dusp4. Aberrant Foxl1 activation inhibited undifferentiated cell growth and survival, while DUSP4 suppressed self-renewal pathways. We therefore uncover an essential role for SALL4 in maintenance of undifferentiated spermatogonial activity and identify regulatory pathways critical for germline stem cell function.

Inhibition of SALL4 reduces tumorigenicity involving epithelial-mesenchymal transition via Wnt/β-catenin pathway in esophageal squamous cell carcinoma.

BackgroundGrowing evidence suggests that SALL4 plays a vital role in tumor progression and metastasis. However, the molecular mechanism of SALL4 promoting esophageal squamous cell carcinoma (ESCC) remains to be elucidated.MethodsThe gene and protein expression profiles- were examined by using quantitative real-time PCR, immunohistochemistry and western blotting. Small hairpin RNA was used to evaluate the role of SALL4 both in cell lines and in animal models. Cell proliferation, apoptosis and invasion were assessed by CCK8, flow cytometry and transwell-matrigel assays. Sphere formation assay was used for cancer stem cell derivation and characterization.ResultsOur study showed that the transcription factor SALL4 was overexpressed in a majority of human ESCC tissues and closely correlated with a poor outcome. We established the lentiviral system using short hairpin RNA to knockdown SALL4 in TE7 and EC109 cells. Silencing of SALL4 inhibited the cell proliferation, induced apoptosis and the G1 phase arrest in cell cycle, decreased the ability of migration/invasion, clonogenicity and stemness in vitro. Besides, down-regulation of SALL4 enhanced the ESCC cells’ sensitivity to cisplatin. Xenograft tumor models showed that silencing of SALL4 decreased the ability to form tumors in vivo. Furthermore, our study demonstrated that SALL4 played a vital role in modulating the stemness of ESCC cells via Wnt/β-catenin signaling pathway and in epithelial-mesenchymal transition.ConclusionsOur results revealed that SALL4 might serve as a functional marker for ESCC cancer stem cell, a crucial marker for prognosis and an attractive candidate for target therapy of ESCC.