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Circular RNAs (circRNAs) exhibit significant functions in diverse malignant tumors, including lung adenocarcinoma (LUAD). In this study, we aimed to elucidate the role of circRNA scm like with four mbt domains 2 (circSFMBT2) in LUAD. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot assay or immunohistochemistry (IHC) assay was performed for quantification of circSFMBT2, microRNA-1305 (miR-1305), spalt like transcription factor 4 (SALL4), proliferating Cell Nuclear Antigen (PCNA) or Ki-67. 5-ethynyl-2'-deoxyuridine (EdU) assay, transwell assay and flow cytometry analysis were applied to analyze cell proliferation, metastasis and apoptosis, respectively. Mouse xenograft model was established to explore the function of circSFMBT2. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to estimate the relationship between miR-1305 and circSFMBT2 or SALL4. CircSFMBT2 was upregulated in LUAD and related to advanced TNM stage and poor prognosis. CircSFMBT2 knockdown suppressed cell proliferation, metastasis, glycolysis and induced apoptosis in LUAD cells in vitro as well as tumor formation in vivo. CircSFMBT2 directly targeted miR-1305, and miR-1305 inhibition reversed circSFMBT2 knockdown-mediated inhibitory effects on LUAD malignant behaviors. SALL4 was the target gene of miR-1305. MiR-1305 overexpression repressed the malignant phenotypes of LUAD cells, while SALL4 enhancement abated the effects. CircSFMBT2 aggravated the progression of LUAD by the miR-1305/SALL4 axis, which might provide a diagnostic and prognostic marker for LUAD. [ABSTRACT FROM AUTHOR]

Background: It has been established that Spalt-Like Transcription Factor 4 (SALL4) promotes Colorectal Cancer (CRC) cell proliferation. Furthermore, Amphiregulin (AREG) is crucially involved in cancer cell proliferation and therapeutic resistance regulation. In this regard, this study aimed to establish whether SALL4 affects the radiosensitization of CRC cells via AREG expression regulation. Methods: Transcriptome sequencing and the Human Transcription Factor Database (HumanTFDB) were used to identify the potential SALL4 targets. The dual-luciferase reporter analysis was used to confirm the SALL4-induced AREG activation. Western Blot (WB) and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were used to examine the effect of X-ray irradiation on SALL4 and AREG expression. The AREG-KD (Knockdown) stable cell lines were created through lentiviral infection. Cell proliferation was tracked using Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (EdU)-incorporation assays. Cell cycle and apoptosis were examined through flow cytometry. The cells were exposed to a controlled X-ray radiation dose (6 Gy) for imaging purposes. Results: SALL4 could bound to the AREG promoter, enhancing AREG expression. Furthermore, irradiation upregulated SALL4 and AREG in CRC cells. Additionally, AREG knockdown in CRC cells led to reduced DNA replication efficiency, suppressed cell proliferation, increased DNA damage, and enhanced G1 phase arrest and apoptosis following irradiation. On the other hand, AREG overexpression reversed the inhibitory effects of SALL4 downregulation on AREG expression. Conclusion: In CRC cells, SALL4 downregulation suppressed AREG expression, regulating CRC cell radiosensitivity via the PI3K-AKT pathway, thus presenting a potential therapeutic pathway for CRC treatment using Radiotherapy (RT). [ABSTRACT FROM AUTHOR]

Abstract Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

Testicular tumors (TTs) are rare in children, posing diagnostic and therapeutic challenges. This retrospective study evaluates the diagnostic and prognostic utility of SALL4 and OCT3/4 in pediatric TTs. We analyzed 18 cases of different types of TTs using immunohistochemistry (IHC) to assess SALL4 (Spalt-like transcription factor 4) and OCT3/4 (Octamer binding transcription factor 3/4) expression. SALL4 was positive in 83.3% of tumors, while OCT3/4 was positive in 38.9% of tumors, with a significantly higher prevalence in patients aged 12–18 years compared to those aged 0–11 years (p = 0.013). Mixed germinal cell tumors were significantly more frequently associated with OCT3/4 (p = 0.003), and a high immunostaining expression for SALL4 was observed primarily in yolk sac tumors and embryonal carcinoma. Our findings suggest that SALL4 and OCT3/4 immunostaining can aid in accurate diagnosis and treatment planning, and underscores the importance of OCT3/4 as a predictive factor in pediatric testicular tumors, highlighting its substantial correlation with tumor type and its impact on treatment response. These markers may guide personalized therapeutic strategies, potentially improving patient outcomes. [ABSTRACT FROM AUTHOR]

Pediatric ovarian tumors exhibit unique diagnostic and therapeutic challenges. This study evaluates the expression of SALL4 and OCT3/4 biomarkers in pediatric ovarian tumors and their associations with tumor subtype, stage, and clinical outcome. A retrospective analysis was conducted on 64 patients under 18 years old, examining demographic data, tumor characteristics, immunohistochemical staining, and clinical outcomes. Our results show that SALL4 was significantly expressed in adenocarcinoma, dysgerminoma (DSG), mixed germ cell tumors (GCTs), and immature teratoma, while OCT3/4 was highly expressed in DSG and mixed GCTs. Both markers are associated with a higher tumor grade and stage, indicating a more aggressive disease. The SALL4 positivity expression was correlated with high alpha fetoprotein (AFP) and lactate dehydrogenase (LDH) levels, while OCT3/4 positivity significantly predicted the risk of subsequent metastasis. The mean progression-free survival (PFS) was notably shorter in patients with positive markers. These findings underscore the diagnostic and prognostic value of SALL4 and OCT3/4 in pediatric ovarian tumors, aligning with previous research and supporting their use in clinical practice for better disease management and patient outcomes. [ABSTRACT FROM AUTHOR]

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10 , which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4 , zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre -mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation. [ABSTRACT FROM AUTHOR]

Abstract Effective therapeutic targets and early diagnosis are major challenges in the treatment of gastrointestinal tract (GIT) cancers. SALL4 is a well-known transcription factor that is involved in organogenesis during embryonic development. Previous studies have revealed that SALL4 regulates cell proliferation, survival, and migration and maintains stem cell function in mature cells. Additionally, SALL4 overexpression is associated with tumorigenesis. Despite its characterization as a biomarker in various cancers, the role of SALL4 in GIT cancers and the underlying mechanisms are unclear. We describe the functions of SALL4 in GIT cancers and discuss its upstream/downstream genes and pathways associated with each cancer. We also consider the possibility of targeting these genes or pathways as potential therapeutic options for GIT cancers.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Effective therapeutic targets and early diagnosis are major challenges in the treatment of gastrointestinal tract (GIT) cancers. SALL4 is a well-known transcription factor that is involved in organogenesis during embryonic development. Previous studies have revealed that SALL4 regulates cell proliferation, survival, and migration and maintains stem cell function in mature cells. Additionally, SALL4 overexpression is associated with tumorigenesis. Despite its characterization as a biomarker in various cancers, the role of SALL4 in GIT cancers and the underlying mechanisms are unclear. We describe the functions of SALL4 in GIT cancers and discuss its upstream/downstream genes and pathways associated with each cancer. We also consider the possibility of targeting these genes or pathways as potential therapeutic options for GIT cancers. [ABSTRACT FROM AUTHOR]

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within themesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes. [ABSTRACT FROM AUTHOR]

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

CD44 and SALL4 are markers of stemness and expressed in cancer stem cells (CSCs). Their deregulated expression was seen in various tumors and has been correlated with clinical severity. We evaluated immunohistochemical expression of CD44 and SALL4 in leukoplakia and oral squamous cell carcinoma (OSCC). CD44 was expressed in all the cases of leukoplakia and Its expression correlated with severity of the dysplasia in leukoplakia but varied with differentiation in OSCC. SALL4 did not express in leukoplakia. Its expression was varied in OSCC cases. We opine that CD44 expression is consistent with the progression of dysplasia in leukoplakia and differentiation in OSCC.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnbl). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF. [ABSTRACT FROM AUTHOR]

Testicular tumors (TTs) are rare in children, posing diagnostic and therapeutic challenges. This retrospective study evaluates the diagnostic and prognostic utility of SALL4 and OCT3/4 in pediatric TTs. We analyzed 18 cases of different types of TTs using immunohistochemistry (IHC) to assess SALL4 (Spalt-like transcription factor 4) and OCT3/4 (Octamer binding transcription factor 3/4) expression. SALL4 was positive in 83.3% of tumors, while OCT3/4 was positive in 38.9% of tumors, with a significantly higher prevalence in patients aged 12–18 years compared to those aged 0–11 years (p = 0.013). Mixed germinal cell tumors were significantly more frequently associated with OCT3/4 (p = 0.003), and a high immunostaining expression for SALL4 was observed primarily in yolk sac tumors and embryonal carcinoma. Our findings suggest that SALL4 and OCT3/4 immunostaining can aid in accurate diagnosis and treatment planning, and underscores the importance of OCT3/4 as a predictive factor in pediatric testicular tumors, highlighting its substantial correlation with tumor type and its impact on treatment response. These markers may guide personalized therapeutic strategies, potentially improving patient outcomes.

Pediatric ovarian tumors exhibit unique diagnostic and therapeutic challenges. This study evaluates the expression of SALL4 and OCT3/4 biomarkers in pediatric ovarian tumors and their associations with tumor subtype, stage, and clinical outcome. A retrospective analysis was conducted on 64 patients under 18 years old, examining demographic data, tumor characteristics, immunohistochemical staining, and clinical outcomes. Our results show that SALL4 was significantly expressed in adenocarcinoma, dysgerminoma (DSG), mixed germ cell tumors (GCTs), and immature teratoma, while OCT3/4 was highly expressed in DSG and mixed GCTs. Both markers are associated with a higher tumor grade and stage, indicating a more aggressive disease. The SALL4 positivity expression was correlated with high alpha fetoprotein (AFP) and lactate dehydrogenase (LDH) levels, while OCT3/4 positivity significantly predicted the risk of subsequent metastasis. The mean progression-free survival (PFS) was notably shorter in patients with positive markers. These findings underscore the diagnostic and prognostic value of SALL4 and OCT3/4 in pediatric ovarian tumors, aligning with previous research and supporting their use in clinical practice for better disease management and patient outcomes.

Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which is an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Abstract Background Spalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown. Methods Analyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression. Results SALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM. Conclusion These findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

Pancreatic ductal adenocarcinoma (PDAC) is marked by molecular heterogeneity and poor prognosis. Among the stemness‐related transcription factors, Spalt‐like Transcription Factor 4 (SALL4) is correlated with unfavorable outcomes; however, its roles in PDAC remain unclear. SALL4high expression defines a PDAC subpopulation characterized by a shortened patient survival. Although SALL4 expression was mostly evaluated in tumor cells, our findings identify this embryonic transcription factor as a new biomarker in PDAC‐derived stroma. Gene expression analysis reveals that the SALL4high PDAC subset is enriched in cancer stem cell properties and stromal enrichment pathways; notably, an interaction with cancer‐associated fibroblasts (CAF) activated by TGF‐β. A particular oncogenic network was unraveled where Netrin‐1 and TGF‐β1 collaborate to induce SALL4 expression in CAF and drive their cancer‐stemness‐promoting functions. A 7‐gene stromal signature related to SALL4high PDAC samples was highlighted and validated by immunochemistry for prognosis and clinical application. This SALL4‐related stroma discriminated pancreatic preinvasive from invasive lesions and was enriched in short‐term survivors. Our results show that SALL4 transcriptional activity controls a molecular network defined by a specific stromal signature that characterizes PDAC invasiveness and worse clinical outcomes.

Abstract Background The mechanism involved in prostate cancer (PCa) metastasis is still poorly understood, and several oncogenes are known to regulate this process. However, the role of spalt‐like transcription factor 4 (SALL4) in PCa metastasis remains unclear. Methods We performed RNA‐sequencing to compare the mRNA expression profiles of seven localized PCa tissues and six metastatic PCa tissues. SALL4 was then identified and compared in the localized PCa and metastatic PCa. Immunohistochemical studies, qRT‐PCR, and Western blot were performed to analyze the expression of SALL4 in PCa patients and cell lines. SALL4 expression and its relevance to clinical traits and prognosis were further explored in the TCGA database and in our 68 clinical samples. Subsequently, we knocked down SALL4 in DU145 and PC3 cells and performed a series of functional assays to explore the effect of SALL4 on PCa progression. Finally, protein levels of SALL4 and core components of the MAPK pathway were measured by Western blot, and cells were treated with PD0325901 to observe proliferation and metastasis. Results Significantly higher expression of SALL4 was found in metastatic PCa than in localized PCa. In addition, high SALL4 expression was significantly associated with high pathological T stage, N stage, Gleason score, and poor disease‐free survival in TCGA database and in our clinical samples. Functional studies indicated that knockdown of SALL4 in DU145 and PC3 inhibited proliferation, migration, and angiogenesis. Furthermore, the ERK and P38 protein phosphorylation significantly reduced after knockdown of SALL4 in DU145 and PC3, indicating the inactivation of the MAPK signaling pathway. Finally, the proliferation and migration ability of DU145 and PC3 cells were significantly decreased after PD0325901 treatment. Conclusions SALL4 predicts unfavorable outcome and is closely associated with PCa progression, suggesting that SALL4 may be a promising prognostic marker and potential therapeutic target for PCa.

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4 , we conditionally inactivated Hk2 , which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds. [Display omitted] • Transcription of glycolytic enzyme genes is upregulated in Sall4 conditional knockout limb buds. • SALL4 binds to many genes encoding glycolytic enzymes. • Levels of glycolytic intermediates are reduced in Sall4 conditional knockout limb buds. • Sall4 restricts glycolysis in limb buds. • Hk2 conditional knockout phenocopies hindlimb defects of Sall4 conditional knockout. [ABSTRACT FROM AUTHOR]

Key Clinical Message: Primary HCO is a rare, aggressive ovarian malignant tumor, morphologically resembling HCC. SALL4 can be adopted to differentiate HCO from HCC. The serum AFP and CA125 rather than HE4 can be employed as possible biomarkers to track treatment and monitor recurrence. We report a case of a postmenopausal woman presenting with lower abdominal pain and vaginal bleeding. She went through a maximal debulking surgery, and the pathological biopsy revealed hepatoid carcinoma of the ovary (HCO). Immunohistochemical assay demonstrates SALL4 as an indicator to differentiate HCO from hepatocellular carcinoma (HCC). [ABSTRACT FROM AUTHOR]

Background: Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. Following a thorough literature search we hypothesize that fascin might contribute for the local migratory behaviour of the odontogenic epithelial cells in tumors and cysts while SALL4 may contribute to stemness property. Thus, the aim of the present study was to evaluate the expression of fascin and SALL4 in histopathological variants of Ameloblastoma, Adenomatoid Odontogenic Tumor (AOT) and various odontogenic cysts namely Odontogenic keratocyst (OKC), Dentigerous cyst (DC), Radicular cyst (RC), and Calcifying odontogenic cyst (COC). Methods: Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was evaluated in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 cases each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results: Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of DC and RC having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. Conclusion: Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Key Clinical Message Primary HCO is a rare, aggressive ovarian malignant tumor, morphologically resembling HCC. SALL4 can be adopted to differentiate HCO from HCC. The serum AFP and CA125 rather than HE4 can be employed as possible biomarkers to track treatment and monitor recurrence. Abstract We report a case of a postmenopausal woman presenting with lower abdominal pain and vaginal bleeding. She went through a maximal debulking surgery, and the pathological biopsy revealed hepatoid carcinoma of the ovary (HCO). Immunohistochemical assay demonstrates SALL4 as an indicator to differentiate HCO from hepatocellular carcinoma (HCC).

Background: Spalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown. Methods: Analyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression. Results: SALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM. Conclusion: These findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is marked by molecular heterogeneity and poor prognosis. Among the stemness‐related transcription factors, Spalt‐like Transcription Factor 4 (SALL4) is correlated with unfavorable outcomes; however, its roles in PDAC remain unclear. SALL4high expression defines a PDAC subpopulation characterized by a shortened patient survival. Although SALL4 expression was mostly evaluated in tumor cells, our findings identify this embryonic transcription factor as a new biomarker in PDAC‐derived stroma. Gene expression analysis reveals that the SALL4high PDAC subset is enriched in cancer stem cell properties and stromal enrichment pathways; notably, an interaction with cancer‐associated fibroblasts (CAF) activated by TGF‐β. A particular oncogenic network was unraveled where Netrin‐1 and TGF‐β1 collaborate to induce SALL4 expression in CAF and drive their cancer‐stemness‐promoting functions. A 7‐gene stromal signature related to SALL4high PDAC samples was highlighted and validated by immunochemistry for prognosis and clinical application. This SALL4‐related stroma discriminated pancreatic preinvasive from invasive lesions and was enriched in short‐term survivors. Our results show that SALL4 transcriptional activity controls a molecular network defined by a specific stromal signature that characterizes PDAC invasiveness and worse clinical outcomes. [ABSTRACT FROM AUTHOR]

Background: The mechanism involved in prostate cancer (PCa) metastasis is still poorly understood, and several oncogenes are known to regulate this process. However, the role of spalt‐like transcription factor 4 (SALL4) in PCa metastasis remains unclear. Methods: We performed RNA‐sequencing to compare the mRNA expression profiles of seven localized PCa tissues and six metastatic PCa tissues. SALL4 was then identified and compared in the localized PCa and metastatic PCa. Immunohistochemical studies, qRT‐PCR, and Western blot were performed to analyze the expression of SALL4 in PCa patients and cell lines. SALL4 expression and its relevance to clinical traits and prognosis were further explored in the TCGA database and in our 68 clinical samples. Subsequently, we knocked down SALL4 in DU145 and PC3 cells and performed a series of functional assays to explore the effect of SALL4 on PCa progression. Finally, protein levels of SALL4 and core components of the MAPK pathway were measured by Western blot, and cells were treated with PD0325901 to observe proliferation and metastasis. Results: Significantly higher expression of SALL4 was found in metastatic PCa than in localized PCa. In addition, high SALL4 expression was significantly associated with high pathological T stage, N stage, Gleason score, and poor disease‐free survival in TCGA database and in our clinical samples. Functional studies indicated that knockdown of SALL4 in DU145 and PC3 inhibited proliferation, migration, and angiogenesis. Furthermore, the ERK and P38 protein phosphorylation significantly reduced after knockdown of SALL4 in DU145 and PC3, indicating the inactivation of the MAPK signaling pathway. Finally, the proliferation and migration ability of DU145 and PC3 cells were significantly decreased after PD0325901 treatment. Conclusions: SALL4 predicts unfavorable outcome and is closely associated with PCa progression, suggesting that SALL4 may be a promising prognostic marker and potential therapeutic target for PCa. [ABSTRACT FROM AUTHOR]

Abstract Background SALL4 and HIWI are involved in the maintenance of self-renewal capacity of stem cells. Several scrutinizes have demonstrated that SALL4 and HIWI play a key role in cancer development. However, the correlation between these genes regarding different clinicopathological features of patients with colorectal cancer (CRC) is still unclear. Methods The expression of SALL4 and HIWI in different clinicopathological features of 46 CRC patients was analyzed using relative comparative real-time PCR. Results mRNA expression levels of SALL4 and HIWI genes were significantly correlated with each other in CRC (P = 0.013, Pearson correlation = 0.364). HIWI expression was notably increased in tumors with overexpression of SALL4 in comparison with other samples. This correlation was significant in non-metastatic CRCs compared to the metastatic tumors and in invaded tumors to the serosa (T3/T4) in comparison with non-invaded tumors (T1/T2). Conclusions Based on the significant association of SALL4 and HIWI in different indices of CRC poor prognosis, it may be concluded that simultaneous expression of these genes is notably contributed to the growth and development of the disease, and therefore, their co-overexpression may be considered for prognosis of aggressive CRCs.

An ectopic pancreas is defined as pancreatic tissue outside its normal location, anatomically separated from the pancreas.The transcription factor pancreas/duodenum homeobox protein 1 (PDX1) is involved in maintaining the pancreas and functions in early pancreatic development, beta cell differentiation, and endocrine non beta cells. Pancreatic transcription factor 1 subunit alpha (PTF1A) affects exocrine cell formation and regulation of acinar cell identity, and is expressed in exocrine cells as a transcription factor. The depletion of SALL4 disrupts self-renewal and induces differentiation.To clarify which of PDX1, PTF1A, or SALL4 determines the difference in Heinrich's classification, we examined the localization and number of positive cells. We analyzed the differential expression of PDX1, PTF1A, and SALL4 in large and small ducts in ectopic pancreas by immunohistochemistry. Results showed that the number of PTF1A-positive cells in large ducts was more widespread in type I than in type II in the gastro-duodenum, and more SALL4-positive cells were noticed in large ducts than in small ducts in the gastro-duodenum of type II. Our results revealed that PTF1A might promote exocrine differentiation in developing the pancreatic tissues, and that those with widespread expression differentiate into exocrine cells.

Yue Zhang,1,* Shuanglin Han,1,* Li Lv,2 Xiaomei Wang,3 Yu Zhu,1 Li Ying1 1Department of Gastroenterology, The Second Hospital of Dalian Medical University, Dalian, Liaoning Province, 116027, People’s Republic of China; 2Department of Pathology, The Second Hospital of Dalian Medical University, Dalian, Liaoning Province, 116027, People’s Republic of China; 3Department of Nuclear Medicine, The Second Hospital of Dalian Medical University, Dalian, Liaoning Province, 116027, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Ying, Department of Gastroenterology, The Second Hospital of Dalian Medical University, No. 467 Zhongshan Road, Shahekou Region, Dalian, Liaoning, 116023, People’s Republic of China, Tel +86-17709870657, Email yingli0209@163.comAbstract: HAC is a rare extrahepatic malignancy histologically resembling hepatocellular carcinoma which can occur in many organs. Especially for cases carrying HAC within the stomach or duodenum, we found that typically HAC only happened in either of them and there was no case exhibiting HAC in both sites. Here, we presented a case having HAC in both sites and to our knowledge, this is the first report presenting HAC in these two distinct organs simultaneously. Firstly, a 57-year-old male was tested for increased serum alpha-fetoprotein level. Following PET-CT images showed strengthened FDG uptake in the gastric antrum and proximal duodenum. Esophagogastroduodenoscopy also revealed an irregular lump at the gastric antrum and a deep ulceration at the duodenum bulb. Next, an immunohistochemistry panel confirmed the diagnosis of HAC. Finally, genetic tests were performed on this patient after the clinical diagnosis of HAC to reveal the molecular etiology.Keywords: HAC, case report, AFP, SALL4, endoscopy, NGS

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods: Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results: Fascin immunopositivity was observed in peripheral ameloblast-like cells, and weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which is an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions: Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

Abstract Background Spalt-like transcription factor 4 (SALL4) and aldehyde dehydrogenase1 family member A1 (ALDH1A1) expressing cells have been characterized as possessing stem cell-like properties known as cancer stem cell marker in serous ovarian carcinoma (SOC). Methods The association between SALL4 and ALDH1A1 was observed based on literature review and bioinformatics tools. Therefore, this study aimed to investigate the association between the co-expression of SALL4/ALDH1A1 proteins and clinicopathological parameters and their prognostic value in SOC patients using immunohistochemical staining on tissue microarrays (TMAs). Furthermore, benign tumors and normal tissue samples were compared with the expression of the tumor tissue samples. Results Increased co-expression of SALL4/ALDH1A1 was found to be significantly associated with the advanced FIGO stage (P = 0.047), and distant metastasis (P = 0.028). The results of Kaplan–Meier survival analysis indicated significant differences between disease- specific survival (DSS; P = 0.034) or progression-free survival (PFS; P = 0.018) and the patients with high and low co-expression of SALL4/ALDH1A1, respectively. Furthermore, high level co-expression of SALL4/ALDH1A1 was a significant predictor of worse DSS and PFS in the univariate analysis. The data also indicated that the co-expression of SALL4/ALDH1A1 was an independent prognostic factor affecting PFS. Moreover, the co-expression of SALL4/ALDH1A1 added prognostic values of DSS in patients with SOC who had grade III versus grade I in multivariate analysis. Conclusions Our data demonstrated that high co-expression of SALL4/ALDH1A1 was found to be significantly associated with tumor aggressiveness and worse DSS or PFS in SOC patients. Therefore, co-expression of SALL4/ALDH1A1 may serve as a potential prognostic biomarker of cancer progression in these cases.

Spalt-Like Transcription Factor 4 (SALL4) is a critical factor for self-renewal ability and pluripotency of stem cells. On the other hand, various reports show tight relation of SALL4 to cancer occurrence and metastasis. SALL4 exerts its effects not only by inducing gene expression but also repressing a large cluster of genes through interaction with various epigenetic modifiers. Due to high expression of SALL4 in cancer cells and its silence in almost all adult tissues, it is an ideal target for cancer therapy. However, targeting SALL4 meets various challenges. SALL4 is a transcription factor and designing appropriate drug to inhibit this intra-nucleus component is challenging. On the other hand, due to lack of our knowledge on structure of the protein and the suitable active sites, it becomes more difficult to reach the appropriate drugs against SALL4. In this review, we have focused on approaches applied yet to target this oncogene and discuss the potential of degrader systems as new therapeutics to target oncogenes. [ABSTRACT FROM AUTHOR]

Background: SALL4 and HIWI are involved in the maintenance of self-renewal capacity of stem cells. Several scrutinizes have demonstrated that SALL4 and HIWI play a key role in cancer development. However, the correlation between these genes regarding different clinicopathological features of patients with colorectal cancer (CRC) is still unclear. Methods: The expression of SALL4 and HIWI in different clinicopathological features of 46 CRC patients was analyzed using relative comparative real-time PCR. Results: mRNA expression levels of SALL4 and HIWI genes were significantly correlated with each other in CRC (P = 0.013, Pearson correlation = 0.364). HIWI expression was notably increased in tumors with overexpression of SALL4 in comparison with other samples. This correlation was significant in non-metastatic CRCs compared to the metastatic tumors and in invaded tumors to the serosa (T3/T4) in comparison with non-invaded tumors (T1/T2). Conclusions: Based on the significant association of SALL4 and HIWI in different indices of CRC poor prognosis, it may be concluded that simultaneous expression of these genes is notably contributed to the growth and development of the disease, and therefore, their co-overexpression may be considered for prognosis of aggressive CRCs. [ABSTRACT FROM AUTHOR]

Background: It has been established that Spalt-Like Transcription Factor 4 (SALL4) promotes Colorectal Cancer (CRC) cell proliferation. Furthermore, Amphiregulin (AREG) is crucially involved in cancer cell proliferation and therapeutic resistance regulation. In this regard, this study aimed to establish whether SALL4 affects the radiosensitization of CRC cells via AREG expression regulation., Methods: Transcriptome sequencing and the Human Transcription Factor Database (HumanTFDB) were used to identify the potential SALL4 targets. The dual-luciferase reporter analysis was used to confirm the SALL4-induced AREG activation. Western Blot (WB) and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were used to examine the effect of X-ray irradiation on SALL4 and AREG expression. The AREG-KD (Knockdown) stable cell lines were created through lentiviral infection. Cell proliferation was tracked using Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU)-incorporation assays. Cell cycle and apoptosis were examined through flow cytometry. The cells were exposed to a controlled X-ray radiation dose (6 Gy) for imaging purposes., Results: SALL4 could bound to the AREG promoter, enhancing AREG expression. Furthermore, irradiation upregulated SALL4 and AREG in CRC cells. Additionally, AREG knockdown in CRC cells led to reduced DNA replication efficiency, suppressed cell proliferation, increased DNA damage, and enhanced G1 phase arrest and apoptosis following irradiation. On the other hand, AREG overexpression reversed the inhibitory effects of SALL4 downregulation on AREG expression., Conclusion: In CRC cells, SALL4 downregulation suppressed AREG expression, regulating CRC cell radiosensitivity via the PI3K-AKT pathway, thus presenting a potential therapeutic pathway for CRC treatment using Radiotherapy (RT)., Competing Interests: The author reports no conflicts of interest in this work., (© 2024 Zhang et al.)

Abstract Background Certain gastric cancers exhibit some primitive phenotypes, which may indicate a high malignancy. In histologically differentiated early gastric cancer (EGC), the presence and the clinicopathological significance of the primitive phenotype remain unclear. Methods Using immunohistochemical staining we detected the expression of three primitive phenotypic markers SALL4, Glypican-3(GPC3), and AFP in whole tissue sections of differentiated EGC (gastrectomy specimens, n = 302). For those cases with primitive phenotypes, we analyzed their clinicopathological features and evaluated whether the criteria for endoscopic resection were met. Results We found that 9.3% (28/302) of all differentiated EGC cases have primitive phenotypes, and most of these cases (25/28) exhibit a histomorphology similar to conventional differentiated EGC. Patients with primitive phenotypes had a deeper invasion, a higher rate of ulcer and lymphatic invasion than cases without primitive phenotype. Moreover, patients with primitive phenotypes displayed a significantly higher frequency of LNM than those without (57.1% vs 8.8%, P

To investigate the genetic etiology of anophthalmia and microphthalmia, we used exome sequencing in a Caucasian female with unilateral microphthalmia and coloboma, bilateral optic nerve hypoplasia, ventricular and atrial septal defects, and growth delays. We found two sequence variants in SALL4 - c.[575C>A], predicting p.(Ala192Glu), that was paternally inherited, and c.[2053G>C], predicting p.(Asp685His), that was maternally inherited. Haploinsufficiency for SALL4 due to nonsense or frameshift mutations has been associated with acro-renal ocular syndrome that is characterized by eye defects including Duane anomaly and coloboma, in addition to radial ray malformations and renal abnormalities. Our report is the first description of structural eye defects associated with two missense variants in SALL4 inherited in trans; the absence of reported findings in both parents suggests that both sequence variants are hypomorphic mutations and that both are needed for the ocular phenotype. SALL4 is expressed in the developing lens and regulates BMP4, leading us to speculate that altered BMP4 expression was responsible for the eye defects, but we could not demonstrate altered BMP4 expression in vitro after using small interfering RNAs (siRNAs) to reduce SALL4 expression. We conclude that SALL4 hypomorphic variants may influence eye development.

Alpha-fetoprotein (AFP)-producing gastric carcinoma is a rare subtype of gastric cancer that produces AFP. As an oncogenic glycoprotein that is normally expressed during gestation, AFP is also used as a biomarker for yolk sac tumors, tumors of gonadal origin, hepatocellular carcinoma, and certain gastric carcinomas. Compared with other gastric cancer subtypes, AFP-producing gastric carcinoma is one of the most aggressive gastric tumors due to its high incidence of early liver and lymph node metastasis. Early metastasis and poor prognosis are attributed to the effect of AFP as a growth factor. Tissue biopsy with immunohistochemical staining is helpful in arriving at the diagnosis and differentiating it from metastatic hepatocellular carcinoma. Although currently there is no standard treatment for AFP-producing gastric carcinoma, surgical resection and chemotherapy have been used.

Cancer stem cells are currently considered an important cause of tumoral heterogeneity, treatment failure and recurrence. SALL4 is a marker of stemness in many types of human cancers. In the present study, the expression of SALL4 was analysed in oral squamous cell carcinomas (OSCCs) using immunohistochemistry. This expression was linked to clinical and pathological characteristics of OSCC patients. Using tissue microarrays of OSCCs, a significant majority of these tumours were shown to positively express SALL4 protein in their cytoplasm and nuclei. This expression positively correlated with tumour grade and tumour location within the oral cavity. Furthermore, SALL4 expression was correlated with aberrant expression of beta-Catenin (AEB) in OSCCs. Morphologic evidence presents significant overlap between cells expressing the two proteins. Given the above, and in light of past evidence of SALL4 modulating the Wingless/Wnt/beta-Catenin pathway in other tumours, this study presents a possible role for the SALL4 protein in OSCC development and progression, and asserts the need for further studies into that role.

Background: Spalt-like transcription factor 4 (SALL4) and aldehyde dehydrogenase1 family member A1 (ALDH1A1) expressing cells have been characterized as possessing stem cell-like properties known as cancer stem cell marker in serous ovarian carcinoma (SOC). Methods: The association between SALL4 and ALDH1A1 was observed based on literature review and bioinformatics tools. Therefore, this study aimed to investigate the association between the co-expression of SALL4/ALDH1A1 proteins and clinicopathological parameters and their prognostic value in SOC patients using immunohistochemical staining on tissue microarrays (TMAs). Furthermore, benign tumors and normal tissue samples were compared with the expression of the tumor tissue samples. Results: Increased co-expression of SALL4/ALDH1A1 was found to be significantly associated with the advanced FIGO stage (P = 0.047), and distant metastasis (P = 0.028). The results of Kaplan–Meier survival analysis indicated significant differences between disease- specific survival (DSS; P = 0.034) or progression-free survival (PFS; P = 0.018) and the patients with high and low co-expression of SALL4/ALDH1A1, respectively. Furthermore, high level co-expression of SALL4/ALDH1A1 was a significant predictor of worse DSS and PFS in the univariate analysis. The data also indicated that the co-expression of SALL4/ALDH1A1 was an independent prognostic factor affecting PFS. Moreover, the co-expression of SALL4/ALDH1A1 added prognostic values of DSS in patients with SOC who had grade III versus grade I in multivariate analysis. Conclusions: Our data demonstrated that high co-expression of SALL4/ALDH1A1 was found to be significantly associated with tumor aggressiveness and worse DSS or PFS in SOC patients. Therefore, co-expression of SALL4/ALDH1A1 may serve as a potential prognostic biomarker of cancer progression in these cases. [ABSTRACT FROM AUTHOR]

Alpha-fetoprotein (AFP)-producing gastric carcinomas (AFPGCs) are relatively rare tumors known to have a poor prognosis and commonly found as advanced lesions. Histologically, AFPGCs have been described as having hepatoid and fetal enteric (enteroblastic) morphology and are associated with conventional adenocarcinomas. Prior studies reported a hepatoid component present only in invasive areas and hypothesized that AFPGCs may develop hepatoid features during the process of tumor invasion. We report three cases of AFP-producing early gastric cancer which had an intramucosal hepatoid component. Immunohistochemistry showed that the hepatoid component was diffusely immunoreactive for SALL4, AFP, arginase-1, and HepPar1, and focally for CDX2 and PDX1. An intramucosal transition between the hepatoid component and conventional intramucosal adenocarcinoma was identified. Two patients also had a coexistent fetal enteric component, which was admixed with a hepatoid component. Although at an early stage one patient subsequently developed liver metastasis and a second patient was suspected of having liver metastasis, these were not biopsy-proven. The latter patient had a previous history of hepatocellular carcinoma (HCC) and SALL4 was used on the HCC to distinguish metastatic/further HCC from a gastric metastatic primary with hepatoid differentiation.

Abstract Background Although 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. Methods Analyses 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. Results SALL4 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. Conclusions Overall, 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.

Abstract Background The stem cell factor SALL4 is reactivated in human cancers. SALL4 plays diverse roles in tumor growth, metastasis, and drug resistance, but its role in tumor metabolism has not been well characterized. Methods The glycolytic levels of gastric cancer cells were detected by glucose uptake, lactate production, lactate dehydrogenase activity, ATP level, and hexokinase activity. QRT-PCR and western blot were used to detect the changes in the expression of glycolytic genes and proteins. The downstream target genes of SALL4 were identified by microarray. The regulation of hexokinase II (HK-2) by SALL4 was analyzed by luciferase reporter assay and chromatin immunoprecipitation assay. Transwell migration assay, matrigel invasion assay, cell counting assay and colony formation assay were used to study the roles of HK-2 regulation by SALL4 in gastric cancer cells in vitro. The effects of SALL4 on glycolysis and gastric cancer progression in vivo were determined by subcutaneous xenograft and peritoneal metastasis tumor models in nude mice. Results SALL4 knockdown inhibited glucose uptake, lactate production, lactate dehydrogenase activity, ATP level and hexokinase activity in gastric cancer cells, and decreased the expression of glycolytic genes and proteins. Microarray analysis showed that SALL4 knockdown affected glycolysis-related pathway. The regulation of HK-2 gene expression by SALL4 was confirmed by luciferase reporter assay and chromatin immunoprecipitation assay. HK-2 knockdown abrogated the promotion of glycolysis by SALL4 in gastric cancer cells, indicating that HK-2 acts as a downstream effector of SALL4. Moreover, HK-2 knockdown reversed the promoting role of SALL4 in gastric cancer cell proliferation, migration and invasion, suggesting that SALL4 drives gastric cancer progression by upregulating HK-2. Conclusions SALL4 promotes gastric cancer progression through HK-2-mediated glycolysis, which reveals a new mechanism for the oncogenic roles of SALL4 in cancer.

Farzaneh Ohadi, Soheila Rahgozar, Elaheh Sadat Ghodousi Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, IranCorrespondence: Soheila RahgozarDepartment of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Hezar Jarib Street, Isfahan 81746-73441, IranTel +98 313 793 2455Fax +98 313 793 2456Email rahgozar@sci.ui.ac.irPurpose: Sal‐like protein 4 transcription factor (SALL4) is a stem cell transcription factor that plays an essential role in the maintenance and self-renewal of embryonic and hematopoietic stem cells, functioning as an oncogene in several cancers. However, the role of SALL4 in the biological behavior of childhood acute lymphoblastic leukemia and its relationship with multidrug resistance and relapse has remained largely unknown.Patients and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to characterize the expression pattern of SALL4 in the bone marrow samples of 43 patients with Philadelphia negative ALL and 18 children in the non-cancer control group. The presence of minimal residual disease was measured a year after the initial therapy using SSCP (single-strand conformation polymorphism). In addition, the correlation between the expression of SALL4 and ABCA3 in relapsed patients was analyzed statistically.Results: Results showed an overexpression of SALL4 in de novo patients compared with the control group (P=0.0001, AUC= 0.93), indicating the importance of this gene in the induction of leukemia. A significant increase in the ABCA3 expression levels was revealed in the relapsed patients, in comparison with the drug-sensitive group (P = 0.0005). The leukemogenetic effect of SALL4 can be related to the effect of this gene on the maintenance of pluripotency in cancer stem cells. Results also suggest that the expression of SALL4 can be considered as a diagnostic marker for pediatric ALL. Moreover, SALL4 expression levels in the minimal residual disease positive (mrd+) ALL group was significantly higher than those in the mrd− group (p=0.0001, AUC= 0.92).Conclusion: These data demonstrate the prognostic impact of SALL4 in childhood ALL. Our findings also indicated a direct correlation between the mRNA expression levels of SALL4 and ABCA3 transporter in the relapsed group of ALL patients (r=0.7). These results describe a possible mechanism by which SALL4 may lead to the development of multidrug resistance.Keywords: childhood acute lymphoblastic leukemia, multidrug resistance, SALL4, ABC transporters, relapse, ABCA3

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

Abstract Extragonadal germ cell tumors (EGGCTs) are uncommon neoplasms, which arise in anatomical locations other than gonads. The pathogenesis of these neoplasms is still poorly understood and it is a matter of debate if they really represent extragondal primary neoplasms or rather extragondal metastasis from occult gonadal neoplasms. The actual observations suggest that EGGCTs represent a unique entity, so their biology and behavior are substantially different from gonadal counterparts. The diagnosis of EGGCTs is often challenging, and differential diagnosis is particularly wide. Nevertheless, a correct diagnosis is essential for the correct management of the patient. We summarize the state of art about EGGCTs, with particular emphasis on diagnosis and prognosis.