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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.

Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2′-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. Significance: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.

AIMS Primary mediastinal germ cell tumours (PMGCTs) are rare mediastinal neoplasms, and their diagnosis can be challenging, owing to small biopsy samples. The aim of this study was to develop a diagnostic algorithm using immunohistochemical staining, with a focus on novel markers, and molecular analysis of isochromosome 12p [i(12p)]. METHODS AND RESULTS Paraffin-embedded tissues of 32 mediastinal tumours were analysed with immunohistochemical staining for sal-like transcription factor 4 (SALL4), Lin-28 homologue A (LIN28), octamer-binding transcription factor 3/4 (OCT3/4), D2-40, cluster of differentiation 117 (CD117), sex-determining region Y-box 17, sex-determining region Y-box 2 (SOX2), cluster of differentiation 30, the β-subunit of human chorionic gonadotropin (β-hCG), GATA-binding protein 3 (GATA3), forkhead box protein A2 (FOXA2), glypican-3 (GPC3), α-fetoprotein (AFP), terminal deoxynucleotidyl transferase (TdT), nuclear protein of the testis (NUT), and pan-cytokeratin. Quantitative real-time polymerase chain reaction was performed to investigate the i(12p) status. Fifteen seminomas, seven teratomas, one yolk sac tumour, one choriocarcinoma and seven mixed PMGCTs were diagnosed. Each entity had different immunohistochemical staining patterns, which helped to distinguish them: OCT3/4, D2-40, CD117 and TdT for seminoma; OCT3/4 and SOX2 for embryonal carcinoma; FOXA2, GPC3 and AFP for yolk sac tumour; and β-hCG and GATA3 for choriocarcinoma. Mature teratomas stained positively for pan-cytokeratin in epithelial components and focally for SALL4, SOX2, GATA3, D2-40, and FOXA2. Furthermore, a NUT carcinoma mimicking a PMGCT was diagnosed, showing strong nuclear SOX2 staining and speckled nuclear NUT staining. i(12p) was detected in 24 of 27 PMGCTs (89%). CONCLUSION A diagnostic algorithm is of great importance for a reliable diagnosis of PMGCT in, usually small, tissue biopsy samples. Therefore, a combination of three to four antibodies to identify the correct histological subtype is usually necessary, in addition to morphological features. The i(12p) status serves as an additional option to indicate a germ cell origin in selected cases.

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.

Summary In addition to hepatoid adenocarcinoma (HAC), gastric adenocarcinoma with enteroblastic differentiation (GAED) and common adenocarcinoma (COM) could also show hepatoid differentiation, which presents a poor prognosis. To elucidate the histogenesis and development of gastric cancer with hepatoid differentiation, we identified 55 cases by histological morphology and a panel of markers, including α-fetoprotein (AFP), Glypican 3 (GPC3) and SALL4, then clinicopathological parameters, pathomorphological characteristics, mucin phenotypes, molecular features, Immunoscore and survival analysis were assessed. A mixture of three types (COM + GAED + HAC) was most commonly observed in the same case, and typical transitions between each histological subtype were frequently seen. Hyaline globule and pink amorphous substance were often present. HER2 was amplified in 21.8% of cases. All the tumors showed intestinal phenotype (69.1%) and mixed gastric/intestinal phenotype (30.9%) and were all defined to chromosomal instable (CIN)/genomically stable (GS) group. Considering that 83.6% cases presented TP53 gene mutation phenotype and 61.8% cases showed ≥10% aberrant E-cadherin expression, the precise molecule classification is ambiguous. Survival analysis showed that patients with high SALL4 expression, high preoperative serum AFP level, or low Immunoscore had a significantly poor overall survival (OS). Moreover, SALL4, HER2, and Immunoscore had an independent influence on OS. In conclusion, we suggest that the development of gastric adenocarcinoma with hepatoid differentiation might a continuously progressive profile: from intestinal-type COM adenocarcinoma to GAED and then HAC. CIN/GS subtypes might be where they belonged. SALL4, HER2, and Immunoscore may be potential therapeutic targets.

NUT carcinoma, also known as NUT midline carcinoma, is an aggressive malignancy mainly affecting the midline structures of younger patients and almost invariably leading to death within a few months of the diagnosis. Morphologically, NUT carcinoma consists of sheets of monomorphous small or medium size cells with scant cytoplasm, commonly featuring areas of abrupt squamous differentiation with keratinization. Immunohistochemistry for NUT protein is sensitive and specific, typically showing a speckled nuclear reactivity, assisting in diagnosis. The molecular background of NUT carcinoma includes the reciprocal translocation t(15;19) leading to expression of the BRD4-NUT fusion transcript with oncogenic properties. Other less common genes may occasionally be fused with NUT not only in NUT carcinoma but also in other soft tissue tumors, highlighting the fact that NUT-rearranged tumors may represent a larger and more diverse family of neoplasms. Thoracic SMARCA4-deficient undifferentiated tumors are aggressive malignancies diagnosed more often in young male smokers, which often lead to death within a few months. SMARCA4-deficient tumors show undifferentiated morphology with occasional hepatoid and rhabdoid features. Immunohistochemically, the hallmark of diagnosis is loss of expression of SMARCA4 (BRG1). Concurrent loss of SMARCA2 expression, as well as expression of one or more stem cell markers SOX2, CD34, or SALL4 is common. Truncating mutations in SMARCA4, a catalytic subunit of the mammalian BAF (SWI/SNF) complex, are the dominant oncogenic molecular event underlying the pathogenesis of these tumors. SMARCA4 deficiency can also be seen as a passenger somatic event in multiple solid neoplasms manifesting as focal dedifferentiation and rhabdoid morphology.

Stemness phenotype is considered as the centerpiece of cancer biology due to its potential in conventional chemo-radiotherapy resistance and tumor recurrence after clinical intervention. This feature in tumor mass belongs to activation of core regulatory stemness factors and different cell signaling pathways in cancer stem cells. We aimed in this study to elucidate contribution of Notch signaling pathway in stemness state of esophageal squamous cell carcinoma (ESCC) through their relevance with stem cell markers SOX2 and SALL4. 50 ESCC tumor and related margin normal tissues were considered and categorized based on SOX2/SALL4 expression pattern, and mRNA levels of Notch signaling genes including ligands, receptors, target genes, and transcriptional coactivator were analyzed in the selected groups using qRT-PCR. Concomitant overexpression of stem cell markers SOX2 and SALL4 in ESCCs upregulated the involved genes in Notch signaling pathway. Upregulation of Notch pathway genes associated with depth of tumor invasion and lymph node metastasis of ESCC. Based on biological function of SOX2 and SALL4 axis in stemness state potential, our results may suggest contribution of Notch signaling pathway in self-renewal capacity of ESCCs, as well as invasion and metastasis of the disease. To the best of our knowledge, this is the first report elucidating the crosstalk between SOX2/SALL4 stemness factors and Notch signaling pathway in cancer research.

Background: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. Methods: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. Results: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. Conclusions: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Rare hepatoid teratomas (HTs) in testicular germ cell tumor patients mimic hepatoid yolk sac tumor (HYST) and hepatocellular carcinoma (HCC). We compared the features of 2 metastatic HTs, 12 HYSTs, and 16 HCCs. The mean ages were 36, 40, and 62.5 years, respectively. The HTs formed sheets of hepatocyte-like cells with macrovesicular fat arranged in vague lobules with intervening fibrous bands containing biliary ductule-like structures and abortive portal triads. HTs lacked basement membrane deposits, with hepatoid cells staining for glypican-3, arginase, and HepPar-1 (2/2), whereas stains for CK19 (2/2) and CK7 (1/2) highlighted ductules and for villin hepatoid cells and ductules (1/2). SALL4 and CDX2 stains were negative (0/2). HYSTs formed nests, trabeculae, cords, and occasional gland-like structures, and most (10/12; 83%) produced intercellular basement membrane. No Mallory-Denk bodies were seen. Stains for SALL4 (100%), glypican-3 (100%), CK19 (88%), CDX2 (88%), and villin (75%) were positive, whereas those for HepPar-1 highlighted rare tumor cells (70%) and for arginase were mostly negative (26%). All HCCs lacked basement membrane deposits, with Mallory-Denk bodies occurring in 50%. Stains for HepPar-1 (100%) and arginase (94%) were positive, glypican-3 infrequent (19%), and SALL4, CK19, villin, and CDX2 negative. In summary, HTs are distinguished from HYST by the formation of ductules and abortive portal tracts, lack of basement membrane deposits, more consistent staining for arginase and HepPar-1, and negativity for SALL4 and CDX2. Contrasting features of HCCs with HYSTs include negativity for SALL4, CK19, and CDX2, frequent Mallory-Denk bodies, and absence of basement membrane deposits.

Cancer stem cells have been implicated angiogenesis of tumor and invasiveness, drug resistance in tumors. Yes-associated protein 1 (YAP) owns carcinogenic roles in various organs, but the role of YAP in cancer stem cells of gastric cancer (GC) remains unclear. In this study, we explored the function and mechanism of YAP in GC cancer stem cells. First, we confirmed that the expression of YAP mRNA and protein in GC tissues was higher than in adjacent tissues by RT-PCR, western blot and immunohistochemistry. Immunofluorescence staining of the GC tissues revealed that the region of YAP expression coincided with the region of expression of the cancer stem cell marker SALL4 but did not overlap with that of the epithelial marker cytokeratin 14 (CK14). Additional research revealed that spherical cells expressed relatively high levels of YAP protein, and YAP overexpression reinforced self-renewal and expression of stem cell markers in the GC cells. Knockdown the expression of YAP reversed this phenomenon. Second, we examined the expression patterns of lipocalin-type prostaglandin D2 synthase (L-PTGDS) and prostaglandin D2 receptor 2 (PTGDR2) in GC tissues and proved that there was negatively correlation between the expression of L-PTGDS and PTGDR2 and YAP in GC tissues. Finally, we confirmed that YAP inhibited the expression of L-PTGDS and PTGDR2 by gain- and loss-of-function experiments. Moreover, the overexpression of L-PTGDS and PTGDR2 suppressed the proliferation and self-renewal induced by YAP in vitro and reversed the pro-tumor effect of YAP in vivo. Our results revealed a novel function of YAP and the mechanism underlying cancer stem cell regulation by YAP.

The existence of a true mixed germ cell-sex cord stromal tumor (MGSCT) of the testis remains controversial. Based on our experience with rare testicular tumors in this spectrum, we sought to perform a detailed clinicopathologic and molecular study of MGCSCT. Eight cases of testicular MGSCT were morphologically reviewed, screened for chromosomal aberrations (using array comparative genomic hybridization (aCGH) and low pass genomic sequencing), and analyzed by next generation sequencing (The Illumina TruSight Tumor 170). Immunohistochemistry for OCT3/4, Nanog, SALL4, DMRT1, and inhibin was performed on the cohort. Clinical data and follow-up were assessed by medical record review. All patients were karyotypically normal men aged 27-74 years (median 41). All tumors had a similar biphasic morphology characterized by various proportions of the sex cord component resembling granulosa cell tumor of adult type and the germ cell component cytomorphologically akin to spermatocytic tumor. Germ cells were haphazardly scattered throughout the tumor or arranged in larger groups, without tubular formation. In 4 cases, atypical mitoses were found within the germ cells. Additionally, in 2 cases there was invasion into the spermatic cord, adjacent hilar soft tissue and into the tumor capsule, which contained both tumor components. Immunohistochemically, focal nuclear expression of DMRT1 was found in the germ cell component in 7/7 analyzable tumors, while SALL4 was positive in 6 cases and negative in one case. All tumors were negative with OCT3/4 and Nanog. The sex cord stromal component had immunoreactivity for inhibin in 7/7 analyzable cases. Four of 8 cases were cytogenetically analyzable: 4/8 by low pass genomic sequencing and 2/8 by aCGH. The results of both methods correlated well, revealing mostly multiple chromosomal losses and gains. One case revealed loss of chromosome 21; 1 case had loss of chromosomes 21 and 22 and partial gain of 22; 1 case had loss of chromosomes 22 and Y, partial loss of X, and gain of chromosomes 20, 5, 8, 9, 12, and 13; and the remaining one gain of chromosomes 20, 3, 6, 8, 2x(9), 11, 2x(12), 13, 14, 18, and 19. Three cases were analyzable by NGS; clinically significant activating mutations of either FGFR3 or HRAS were not detected in any case. Follow-up was available for 4 patients (12, 24, 84, and 288 months) and was uneventful in all 4 cases. The identification of extratesticular invasion of both the germ cell and sex cord stromal components, the DMRT1 expression, and the presence of atypical mitoses in germ cells argue for the neoplastic nature of the germ cell component. The molecular genetic study revealing multiple chromosomal losses and gains in a subset of the cases provides the first evidence that molecular abnormalities occur in testicular MGSCT. Multiple chromosomal aneuploidies, namely, recurrent losses of chromosomes 21 and 22 and gains of 8, 9, 12, 13, and 20, indicate that the germ cell component might be related to the morphologically similar spermatocytic tumor, which is characterized by extensive aneuploidies including recurrent gains of chromosomes 9 and 20 and loss of chromosome 7. In summary, our data support that rare examples of true MGSCT of the testis do exist and they represent a distinct tumor entity with admixed adult-type granulosa cell tumor and spermatocytic tumor components.

The homeobox transcription factor MEIS1 is involved in cell fate decision, stem cells properties, gastrointestinal (GI) tract development, and progression of several malignancies such as esophageal squamous cell carcinoma (ESCC). Increasing evidences suggest the crosstalk between MEIS1 and cell signaling pathways. Therefore, our aim in present study was to investigate the probable linkage of MEIS1 expression with key genes of different cell signaling pathways in ESCC tumorigenesis, and their correlation with clinicopathological feature of the patients. The gene expression profiling of MEIS1 and different cell signaling genes including SALL4, SIZN1, and HEY1 (stemness state, BMP, and NOTCH signaling pathways, respectively) was performed using quantitative real-time reverse transcription polymerase chain reaction (PCR) in fresh tumoral compared to margin normal tissues of 50 treatment-naive ESCC samples. The mRNA expression of MEIS1/SIZN1, SIZN1/HEY1, and SIZN1/SALL4 were significantly associated to each other (P

The aim of the present study was to investigate the expression of spalt like transcription factor 4 (SALL4) in the three most common types of renal cell carcinomas (RCC) [clear cell RCC (ccRCC), papillary renal cell carcinoma (pRCC) and chromophobe RCC (chRCC)], and the association with the overall survival (OS) of patients. The Cancer Genome Atlas (TCGA) database and RCC samples were used to investigate the expression levels of the SALL4 gene and its association with the OS in the three types of RCC based on the analysis of the transcriptome, copy number and survival data. It was found that SALL4 was highly expressed in ccRCC and pRCC tumor tissue, and low mRNA expression level of SALL4 indicated a prolonged survival in both ccRCC and pRCC. This mRNA expression level was associated with pathological Tumor-Node-Metastasis stage, M and T stages in both ccRCC and pRCC. The analysis of the enriched pathway results suggested that SALL4 may act via translation initiation, and that the related genes promoted the progression of RCC. Moreover, the high expression level of SALL4 was detected in RCC samples and serum from patients. It was demonstrated that SALL4 promotes increased viability in RCC cells. Therefore, the present results suggest that SALL4 may be a sensitive and specific cancer biomarker in ccRCC and pRCC. Furthermore, targeting of SALL4 may improve RCC therapy and prolong the survival of patients with ccRCC or pRCC.

SALL4 is a zinc finger transcription factor that exerts its physiological role during embryo-fetal development. Analyses of SALL4 expression have shown its oncogenic role in precursor B-cell lymphoblastic lymphoma, acute and chronic myeloid leukemia, gastrointestinal, breast, and lung cancers. The aim of this study was to determine the immunohistochemical profile of SALL4 in pediatric yolk sac tumors (YSTs). Immunohistochemistry detection of SALL4 was performed in 22 cases of pediatric YSTs and 10 mature teratomas. The percentage of tumor cells stained was scored as 0, 1+ (1–30% cells), 2+ (31–60%), 3+ (61–90%), and 4+ (> 90%). To compare its sensitivity and specificity with Glypican-3 and α-fetoprotein (AFP), we also stained tumors from these cases for Glypican-3 and AFP. In contrast to AFP and glypican-3, SALL4 staining in more than 90% of the tumor cells was seen in all 22 pediatric YSTs (100% sensitivity) (P

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1. (C) 2019 The Authors. Published by Elsevier Inc.

Uterine yolk sac tumors have gained increased recognition in recent years. The current study is a multi-faceted examination of yolk sac tumor-like phenotypes in endometrial tumors, based on an analysis of 3 groups of uterine tumors: Group 1: 9 endometrial tumors that had been classified as yolk sac tumor, or as having a yolk sac tumor component, were assessed with a 35-marker immunohistochemical panel, with the goal of defining their immunophenotypic spectrum; Group 2, comprised of 70 endometrial carcinomas of various histotypes, were analyzed for their expression of SALL4, Glypican-3, and AFP, to assess the specificity of these markers for yolk sac tumors relative to endometrial carcinomas; Group 3, comprised of 626 archived cases of endometrial carcinoma/carcinosarcoma, reviewed to define the frequency of yolk sac tumor-like morphology therein. Yolk sac tumor areas in the Group 1 cases were consistently immunoreactive for SALL4 and Glypican-3; variably positive for AFP (89%), Villin (89%), PLAP (78%), 34βE12 (67%), CAM 5.2 (62.5%), EMA (56%), CD117 (50%), p16 (50%), CDX2 (44%), p53 (44% aberrant), MOC31 (37.5%), CK7 (33%), GATA3 (33%), CK5 (25%), and PAX8 (11%); and were negative for CD30, Napsin A, OCT4, estrogen, androgen, and progesterone receptors. 29 (41%) of the 70 group-2 cases expressed at least one of the 3 markers, and 96% of the positive cases was a high-grade histotype. Glypican-3, SALL4, and AFP were positive in 30, 20, and 2.8% of group-2 cases respectively; however, co-expression of any 2, or all 3 markers was uncommon (

Choriocarcinoma stem-like cells (CSLCs) might be at the origin of choriocarcinoma development associated with drug resistance or relapse. Spalt-like transcription factor 4 (SALL4), which is considered to be a stemness-related gene, can be regulated by miRNAs. In this study, SALL4 result is associated with progression-free survival of choriocarcinoma patients and CSLC’s stemness characteristics. In addition, it could be downregulated by miR-497-5p by direct binding. miR-497-5p silencing by hypermethylation promoted malignant CSLC phenotype in vitro and in vivo. Furthermore, increased DNA methyltransferases (DNMTs) by SALL4 upregulation inhibited miR-497-5p expression via hypermethylation promotion. SALL4 appeared to be a key factor in promoting stemness phenotype of choriocarcinoma. Silencing miR-497-5p and SALL4 promotes choriocarcinoma progression and forms a feedback loop with DNMT-mediated epigenetic regulation, playing a crucial role in stemness maintenance in choriocarcinoma.

Background Both gastric adenocarcinoma with primitive enterocyte phenotype (GAPEP) (including hepatoid adenocarcinoma) and alpha-fetoprotein (AFP)-producing gastric adenocarcinoma have poor prognoses. However, the value of the serum AFP test and AFP/glypican-3 (GPC3)/spalt-like transcription factor 4 (SALL4) immunohistochemistry is still not clear, and these two methods have not yet been thoroughly compared. Methods We collected 421 consecutive non-neoadjuvant surgically or endoscopically resected gastric adenocarcinoma patients with serum AFP results before surgery (group A). We divided these cases into serum AFP-high (sAFP-H) and serum AFP-normal (sAFP-N) by serum AFP levels, and into GAPEP (expressing AFP, GPC3, or SALL4) and non-GAPEP (nGAPEP) by AFP/GPC3/SALL4 immunohistochemistry results. We also collected 12 non-resected gastric adenocarcinoma patients with serum AFP ≥ 7 ng/mL before treatment (group B). We analyzed these patients’ clinicopathological characteristics and prognoses. Results Seventeen (4.04%) patients in group A were sAFP-H. These patients were younger and mainly had tubular adenocarcinoma with later pT (P = 0.014) and pN (P = 0.047) categories and more lymphovascular invasion (P P = 0.008), and metastases or recurrence (P P = 0.001). Most group B patients with elevated serum AFP (especially > 1000 ng/mL) had simultaneous metastases, mainly liver metastases. Both the serological method and immunohistochemical method were useful for predicting prognosis (AUC sAFP = 0.625, AUC A/G/S-IHC = 0.723, z statistic = 1.726, P = 0.084). The serum AFP level (especially > 1000 ng/mL) is more specific (100%), and immunohistochemistry is more sensitive (50%). Conclusion Both the serum AFP level and immunohistochemical expression of AFP/GPC3/SALL4 can be used to indicate a poor prognosis for gastric adenocarcinoma.

Background Cancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression. Methods Fifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture. Results T cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses. Conclusions The IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered.

SALL4 is a zinc finger transcription factor that belongs to the spalt-like (SALL) gene family. It plays important roles in the maintenance of self-renewal and pluripotency of embryonic stem cells, and its expression is repressed in most adult organs. SALL4 re-expression has been observed in different types of human cancers, and dysregulation of SALL4 contributes to the pathogenesis, metastasis, and even drug resistance of multiple cancer types. Surprisingly, little is known regarding how SALL4 expression is controlled, but recently microRNAs (miRNAs) have emerged as important regulators of SALL4. Due to the ability of regulating targets differentially in specific tissues, and recent advances in systemic and organ specific miRNA delivery mechanisms, miRNAs have emerged as promising therapeutic targets for cancer treatment. In this review, we summarize current knowledge of the interaction between SALL4 and miRNAs in mammalian development and cancer, paying particular attention to the emerging roles of the Let-7/Lin28 axis. In addition, we discuss the therapeutic prospects of targeting SALL4 using miRNA-based strategies, with a focus on the Let-7/LIN28 axis.

So-called primary yolk sac tumors of the vulva are very rare and often have an aggressive disease course. Their molecular features have not been previously characterized. There is also a well-documented group of SMARCB1 (INI-1)-deficient vulvar neoplasms, which includes proximal-type epithelioid sarcoma and myoepithelial carcinoma. Until now, "vulvar yolk sac tumors" and SMARCB1-deficient neoplasms were considered unrelated diseases. After reviewing an index case of a vulvar yolk sac tumor with loss of SMARCB1 by immunohistochemistry, we retrospectively identified 2 additional cases diagnosed as vulvar yolk sac tumors. Patient ages were 34, 32, and 25 years old, and 2 tumors were associated with a pregnancy. All 3 cases showed morphology typical of a yolk sac tumor, and by immunohistochemistry all were positive for SALL4, glypican-3, keratins, and lacked CD34 positivity. All tumors also demonstrated loss of SMARCB1 in tumor cells. Targeted molecular profiling was performed in 2 cases and identified 2 copy deletion of SMARCB1, without genomic alterations typically seen in gonadal yolk sac tumors. In the third case, isochromosome 12p was not identified by fluorescence in situ hybridization. All 3 patients had either local recurrences or distant metastases, and 2 died of disease. One patient had progressive disease while receiving the enhancer of zeste homolog 2 inhibitor tazemetostat. Overall, these findings suggest that vulvar tumors with pure yolk sac-like morphology may represent morphologic variants of SMARCB1-deficient tumors and not veritable germ cell neoplasia. This potential reclassification may have both prognostic and treatment implications and warrants study of additional extragonadal yolk sac tumors.

MicroRNAs (miRNAs or miRs) have been found to participate in the development and malignant progression of human cancers by negatively mediating the expression of their target genes. Recently, miR‑33b has been reported to be involved in multiple types of human cancer, including hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms of miR‑33b in HCC cell growth and metastasis remain largely unclear. In the present study, RT-qPCR revealed that miR‑33b was significantly downregulated in HCC tissues compared to their matched adjacent normal tissues. Moreover, the miR‑33b level was significantly lower in advanced-stage HCC (stages T3-T4) compared to early-stage HCC (stages T1-T2). Furthermore, it was also downregulated in the HCC cell lines, LH86, HepG2, LMH and PLHC-1, when compared with the THLE-3 normal human liver cells. We further demonstrated that the overexpression of miR‑33b led to a significant decrease in the proliferation, migration and invasion of HepG2 and LH86 cells. Luciferase reporter assay identified Sal-like protein 4 (SALL4) as a target gene of miR‑33b, and its protein expression was negatively regulated by miR‑33b in HepG2 and LH86 cells. Moreover, the restoration of SALL4 expression markedly reversed the inhibitory effect of miR‑33b overexpression on the proliferation, migration and invasion of HepG2 and LH86 cells, indicating that SALL4 is involved in miR‑33b-mediated malignant phenotypes of HCC cells. Furthermore, we found that SALL4 was significantly upregulated in HCC tissues compared to their matched adjacent normal tissues, and its increased expression was significantly associated with the advanced malignancy of HCC. Moreover, SALL4 was also upregulated in HCC cell lines compared to the THLE-3 normal human liver cells. Finally, we found that the SALL4 expression inversely correlated with the miR‑33b level in HCC tissues. On the whole, the findings of our study demonstrate that miR‑33b suppresses the proliferation and metastasis of HCC cells through the inhibition of SALL4 expression. Therefore, miR‑33b/SALL4 may become a potential therapeutic target for the treatment of HCC.

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 P = 0.001, HR 6.977, 95% CI: 2.199–22.138). Interestingly, we found 2 cases with primitive phenotypes developed LNM, and they both met the expanded indications of endoscopic resection for differentiated EGC. Conclusions A small number of differentiated EGC have primitive phenotypes, which were closely related to LNM and were an independent risk factor for LNM. Given its highly aggressive behavior, differentiated EGC with primitive phenotypes should be evaluated with stricter criteria before endoscopic resection, or considered to give an additional surgical operation after endoscopic resection.

Germline inactivating mutations in SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene encoding for BRG1 (Brahma related gene-1) are the molecular drivers in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) and in malignant rhabdoid tumors (MRT) that occur in the context of rhabdoid tumor predisposition syndrome-type 2. Somatic SMARCA4 mutations and/or loss of BRG1 have been identified in a variety of adult-onset epithelial and mesenchymal neoplasms. Among thoracic tumors, these include subsets of smoking-related non-small cell lung carcinoma (NSCLC) and a relatively rare, newly recognised tumor entity: thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Less than 100 cases of SMARCA4-UT have been reported to date. They present as large compressive and infiltrative mediastinal, lung and/or pleural masses in middle-aged male smokers. They are undifferentiated tumors composed of sheets of small/epithelioid and/or rhabdoid tumor cells variably expressing epithelial markers and consistently showing loss of BRG1 and the closely related protein, Brahma (BRM). Frequent expression of stem cell markers (SOX2, CD34, SALL4) is noted. Despite gene expression profiles similar to MRTs and SCCOHT, they show striking genomic overlap with SMARCA4-mutant NSCLC with frequent TP53, STK11, KEAP1, and KRAS mutations, high tumor mutation burden (TMB), and presence of smoking related molecular signatures in tumor cells. SMARCA4-UT show uniformly poor survival and are irresponsive to conventional therapies. Immunotherapy responses are variable but promising, although PDL1 expression appears to be of poor predictive value. Drugs exploiting genetic and epigenetic mechanisms of SMARCA4 antagonism hold promise for future targeted therapies.

DICER1 syndrome is characterized by a unique combination of features and a growing list of associated rare tumors. Traditionally, gonadal or extra-gonadal teratomas have not been considered part of this spectrum, with only rare DICER1-related teratoid neoplasms recently reported. Besides, their methylation profiles remain elusive. We report two DICER1-associated malignancies involving the lumbar spine of a 22-year-old man (case 1) and the pelvic cavity of a 14-year-old girl (case 2). Both tumors exhibited teratoma-like features with a high-grade malignant somatic component, including rhabdomyosarcomatous elements for case 1 and a malignant neuroectodermal neoplasm with features of an embryonal tumor with multilayered rosettes (ETMR) for case 2. Both tumors showed strong SALL4 expression and H3K27me3 loss by immunohistochemistry. Next-generation sequencing studies confirmed biallelic DICER1 mutations with additional pathogenic missense mutations in KRAS (case 1) and CTNNB1 (case 2). The methylation profile of case 1 clustered with DICER1-associated sarcomas, whereas case 2 classified as an ETMR (albeit low raw and calibrated score). In conclusion, we report two DICER1-related malignancies with teratoma-like features, further expanding their morphologic spectrum and highlighting the multipotentiality of their presumed cell of origin. Notably, we describe the first ETMR identified outside the CNS with a documented DICER1 biallelic inactivation. Our findings also highlight the potential role of other molecular alterations such as KRAS and CTNNB1 mutations in defining the phenotype of embryonal and primitive DICER1-associated neoplasms, a notion that deserves further studies.

Background In acute myeloid leukemia (AML), many genes have been studied as prognostic markers. SALL4 is expressed constitutively in human leukemia cell lines and primary AML cells. BMI-1 is expressed highly in purified hematopoietic stem cells (HSCs), and its expression declines with differentiation. Objective To study the expression levels of SALL4 and BMI-1 and their clinical significance in patients with AML. Methods The study was performed with 60 patients newly diagnosed with AML and 50 control individuals. SALL4 and BMI-1 expression detection were performed using real-time polymerase chain reaction (PCR). Results The expression of SALL4 and BMI-1 was significantly higher in cases of AML and showed a strong association with failure to achieve complete remission (CR) or with relapse (P = .02, P = .03, respectively). In multivariate analysis, these genes were the most powerful independent predictors of poor prognosis (P = .01 for SALL4, P = .02 for BMI-1). Conclusion SALL4 and BMI-1 are significant prognostic factors in AML and could be strong targets for novel types of therapy.

SALL4 is overexpressed in many cancers and is found to be involved in tumorigenesis and tumor progression. However, the function of SALL4 in cervical cancer remains unknown. Here, we showed that the expression of SALL4 was gradually increased from normal cervical tissue to high‐grade squamous intraepithelial lesions and then to squamous cervical carcinoma. SALL4 was upregulated or downregulated in cervical cancer cells by stably transfecting a SALL4‐expressing plasmid or a shRNA plasmid targeting SALL4, respectively. In vitro, cell growth curves and MTT (3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assays showed that SALL4 promoted the cell proliferation of cervical cancer cells. In vivo, xenograft experiments verified that SALL4 enhanced the tumor formation of cervical cancer cells in female BALB/c Nude mice. Cell cycle analysis by fluorescence‐activated cell sorting found that SALL4 accelerates cell cycle transition from the G0/G1 phase to the S phase. TOP/FOP‐Flash reporter assay revealed that SALL4 significantly upregulates the activity of Wnt/β‐catenin pathway. Western blotting showed that the expression levels of β‐catenin and important downstream genes, including c‐Myc and cyclin D1, were increased by SALL4 in cervical cancer cells. Furthermore, dual‐luciferase reporter and chromatin immunoprecipitation assays confirmed that SALL4 transcriptionally activated CTNNB1 by physically interacting with its promoters. Taken together, The results of this study demonstrated that SALL4 may promote cell proliferation and tumor formation of cervical cancer cells by upregulating the activity of the Wnt/β‐catenin signaling pathway by directly binding to the CTNNB1 promoter and trans‐activating CTNNB1.

Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) and have been detected in 7-17% of Philadelphia chromosome-negative BCP-ALL cases. We selected SALL4 and ID2 as potential Z-fusion-specific transcriptional targets that might lead to the differentiation disorder of Z-fusion-positive ALL. The introduction of EP300-ZNF384 and SYNRG-ZNF384 induced the expression of these genes. Z-fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild-Z. Furthermore, GST pull-down assay revealed that Z-fusion proteins associated more strongly with EP300 than Wild-Z. Coexpression of EP300 specifically enhanced the transcriptional activities of Z-fusion proteins. We propose the increased EP300 binding of Z-fusion proteins as a mechanism for their increased transcriptional activities.

Radiotherapy is the mainstay and primary curative treatment modality in nasopharyngeal carcinoma (NPC), whose efficacy is limited by the development of intrinsic and acquired radioresistance. Thus, deciphering new molecular targets and pathways is essential for enhancing the radiosensitivity of NPC. SALL4 is a vital factor in the development and prognosis of various cancers, but its role in radioresistance remains elusive. This study aimed to explore the association of SALL4 expression with radioresistance of NPC. It was revealed that SALL4 expression was closely correlated with advanced T classification of NPC patients. Inhibition of SALL4 reduced proliferation and sensitized cells to radiation both in vitro and in vivo. Furthermore, SALL4 silencing increased radiation‐induced DNA damage, apoptosis, and G2/M arrest in CNE2 and CNE2R cells. Moreover, knockdown of SALL4 impaired the expression of p‐ATM, p‐Chk2, p‐p53, and anti‐apoptosis protein Bcl‐2, while pro‐apoptosis protein was upregulated. These findings indicate that SALL4 could induce radioresistance via ATM/Chk2/p53 pathway and its downstream proteins related to apoptosis. Targeting SALL4 might be a promising approach for the development of novel radiosensitizing therapeutic agents for radioresistant NPC patients.

Background/Aims Sorafenib remains the only approved molecular targeted agent for hepatocellular carcinoma (HCC); however, reliable biomarkers that predict its efficacy are still lacking. The aim of this study was to explore whether cancer stem cell (CSC) markers have a predictive role with regard to the sorafenib response in HCC patients. Methods We enrolled 47 patients with HCC for whom tumor samples obtained before starting sorafenib treatment were available. RNA was extracted from formalin-fixed, paraffin-embedded samples, and real-time polymerase chain reaction was used to quantify mRNA expression of the CSC genes EpCAM, CD13, CK8, CD24, CD44, CD90, CD133, SALL4, ALDH1A1, ALB, and AFP . Results Of 47 patients, 14.9% and 74.5% had vascular invasion and extrahepatic spread, respectively. Patients with low CD133 expression tended to have longer progression-free survival (PFS) than those with high CD133 expression (5.5 months vs 4.0 months), although without statistical significance. The expression levels of other markers were not associated with PFS. When examining markers in combination, patients with high CD133 and CD90 expression had shorter PFS rates than those with low expression (2.7 months vs 5.5 months; p=0.04). Patients with low CD133 and EpCAM expression demonstrated better PFS than those with high expression (7.0 months vs 4.2 months; p=0.04). Multivariable analysis indicated that an Eastern Cooperative Oncology Group performance status score of 1 and high CD133/CD90 expression were significantly associated with shorter PFS. Conclusions Overexpression of the CSC markers CD133 and CD90 in HCC was associated with poorer response to sorafenib. These two genes may serve as predictive biomarkers for sorafenib therapy.

Adult neuroblastoma is an extremely infrequent neoplasm, usually occurring in the adrenal medulla or in the paraspinal sympathetic ganglia, as its childhood counterpart. We report a very unusual case of a Schwannian stroma-poor adult neuroblastoma of inguinal location, showing aberrant expression of germ cell markers: SALL4 and OCT4. This aberrant marker expression, the unusual positivity for NKX2.2 and the very scattered (instead of diffuse strong) PHOX2B expression, complicated the initial diagnosis. In this case, the posttreatment histological evaluation revealed the neuroblastic nature of the lesion. Neuroblastoma maturation after treatment is an unusual finding in adults, and in this case, added an important clue for the final diagnosis. Germs cells markers expression in neuroblastoma is an interesting feature to explore and may define a subset of neuroblastomas with a different biological nature.

We have recently characterized cancer stem cell (CSC) subpopulations in different types of cancer. This study aimed to identify and characterize CSCs within metastatic melanoma (MM) to the brain. 3, 3-diaminobenzidine (DAB) immunohistochemical (IHC) staining of ten samples of MM to the brain demonstrated the expression of the embryonic stem cell (ESC) markers OCT4, NANOG, SALL4, SOX2 and pSTAT3. Protein expression of all five ESC markers except SALL4 were confirmed by Western blotting on five samples and transcriptional activation of all five markers was demonstrated using NanoString mRNA analysis on four samples. Immunofluorescence IHC staining suggested the presence of CSCs that stained for OCT4, SALL4, SOX2 or NANOG. Some of these cells also stained for Melan-A. Also, a pSTAT3+/CD34+ primitive subpopulation was detected on the endothelium of microvessels. These CSCs may be a novel therapeutic target for MM to the brain.

Breast cancer is the most prevalent cancers worldwide and causes a significant amount of deaths annually. Spalt-like transcription factor 4 is known as a transcription factor, which has an important role in the proliferation of cancerous cells. Small interfering RNA (siRNA) is a short-chain molecule of 20 to 25 nucleotides that protrude on two sides of the 3', two nucleotides. In this study, using a specific sequence of siRNA against the sequence of this gene, its activity is investigated in the cell line of breast cancer. The breast cancer cells (MCF-7) were cultured and then, using a specific anti-sal-like 4 (SALL4) siRNA, their toxic doses were determined. Then, the gene is transfected into the cell. Proliferation and expression of the SALL4 and BCL-2 gene were measured using the real-time polymerase chain reaction method. The specific concentration of siRNA IC50 of the SALL4 gene was 40.35 nmole. Gene expression results indicated that the expression of the Bcl-2 gene in the siRNA group was significantly reduced ( P < 0.05). SiRNA can increase the apoptosis of breast cancer cells by reducing the gene expression of SALL4 gene and Bcl-2; it can be used as a novel targeted therapy. This strategy, in addition to increasing the specificity of the drug, also reduces the side effects when compared with conventional chemotherapy.

Accumulating evidence suggested that many long noncoding RNAs (lncRNAs) were widely involved in the development and progression of non-small cell lung cancer (NSCLC). However, the roles of lncRNA homeobox A11 antisense (HOXA11-AS) and its underlying mechanism in NSCLC remains largely unknown. The expression levels of HOXA11-AS, miR-3619-5p and sal-like protein 4 (SALL4) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot analysis was used to measure the protein levels of hexokinase II (HK2) and SALL4. Cell proliferation, apoptosis, migration and invasion were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and transwell assay, respectively. The glucose consumption and lactate production were measured using glucose assay kit and lactate assay kit, respectively. The potential binding sites between miR-3619-5p and HOXA11-AS or SALL4 were predicted by online software and verified by luciferase report assay. A xenograft tumor model was established to confirm the function of HOXA11-AS in NSCLC in vivo. HOXA11-AS and SALL4 were upregulated while miR-3619-5p was downregulated in NSCLC tissues and cells. HOXA11-AS knockdown suppressed cell proliferation, migration, invasion, and glycolysis but promoted apoptosis in NSCLC cells. Moreover, miR-3619-5p could directly bind to HOXA11-AS and its inhibition attenuated the inhibitory effect of HOXA11-AS knockdown on progression of NSCLC cells. Furthermore, SALL4 was a direct target of miR-3619-5p and its overexpression reversed the anti-tumor role of miR-3619-5p in NSCLC cells. Besides, HOXA11-AS modulated SALL4 expression via sponging miR-3619-5p. Additionally, silencing HOXA11-AS inhibited tumor growth though upregulating miR-3619-5p and downregulating SALL4. Collectively, HOXA11-AS knockdown inhibited the progression of NSCLC by regulating miR-3619-5p/SALL4 axis, which might offer a novel avenue for interpreting the mechanism of NSCLC development.

Balancing mRNA nuclear export kinetics with its nuclear decay is critical for mRNA homeostasis control. How this equilibrium is aberrantly disrupted in esophageal cancer to acquire cancer stem cell properties remains unclear. Here we find that the RNA-binding protein interleukin enhancer binding factor 2 (ILF2) is robustly upregulated by nicotine, a major chemical component of tobacco smoke, via activation of JAK2/STAT3 signaling and significantly correlates with poor prognosis in heavy-smoking patients with esophageal cancer. ILF2 bound the THO complex protein THOC4 as a regulatory cofactor to induce selective interactions with pluripotency transcription factor mRNAs to promote their assembly into export-competent messenger ribonucleoprotein complexes. ILF2 facilitated nuclear mRNA export and inhibited hMTR4-mediated exosomal degradation to promote stabilization and expression of SOX2, NANOG, and SALL4, resulting in enhanced stemness and tumor-initiating capacity of esophageal cancer cells. Importantly, inducible depletion of ILF2 significantly increased the therapeutic efficiency of cisplatin and abrogated nicotine-induced chemoresistance in vitro and in vivo. These findings reveal a novel role of ILF2 in nuclear mRNA export and maintenance of cancer stem cells and open new avenues to overcome smoking-mediated chemoresistance in esophageal cancer. Significance: This study defines a previously uncharacterized role of nicotine-regulated ILF2 in facilitating nuclear mRNA export to promote cancer stemness, suggesting a potential therapeutic strategy against nicotine-induced chemoresistance in esophageal cancer.

Objective: We aimed to identify the expression of Sal-like 4 (SALL4) in breast cancer tissues and to explore the role of this gene in the carcinogenesis of breast cancer cells. Methods: A total of 62 paired breast cancer and noncancerous tissue samples were obtained from patients with breast cancer. SALL4 expression patterns and their association with clinicopathological characteristics were investigated by qRT-PCR, western blotting, and immunochemistry in breast cancer tissues. After the knockdown of SALL4 by short hairpin RNAs (shRNAs), the proliferative, invasive, and apoptotic abilities of MDA-MB-435 and MDA-MB-468 cells (breast cancer cell lines) were measured by colony formation and CCK-8 assays, wound healing and transwell assays, and flow cytometry, respectively. Results: SALL4 expression was higher in breast cancer tissues than that in the paired noncancerous tissues, and increased SALL4 expression in tumor tissues was closely related to tumor size and lymphatic metastasis. Furthermore, functional experiments revealed that SALL4 knockdown inhibited the cell proliferation, induced cell cycle arrest in G0/G1phase and apoptosis, and decreased the ability of migration and invasion in breast cancer cells. Additionally, our study first demonstrated that SALL4 played a critical role in modulating the tumorigenicity of breast cancer cells via the WNT/β-catenin signaling pathway. Conclusions: Our results suggest that the expression of SALL4 is upregulated in breast cancer, and this upregulation is involved in the regulation of cell growth, invasion, and apoptosis. Hence, SALL4 may be a promising target for diagnosis and therapy in patients with breast cancer.

Gastric adenocarcinoma with enteroblastic differentiation (GAED) is rare, highly malignant, and has higher vascular invasion and metastasis rates than conventional differentiated gastric cancer (CDGC). We report two cases of GAED that underwent curative resection by endoscopic submucosal dissection (ESD). Case 1 was an 82-year-old man with an elevated lesion in the gastric cardia. Biopsy revealed well-differentiated tubular adenocarcinoma. Pathological diagnosis of the ESD specimen revealed intramucosal gastric cancer without lymphovascular invasion (LVI). Although the surface layer of the lesion showed well and moderately differentiated tubular adenocarcinoma, clear cytoplasmic cancer cells positive for Sal-like protein-4 (SALL4) and Glypican-3 were found in a part of the deep layer. Therefore, GAED was diagnosed as present in a part of the whole lesion and covered with CDGC. Case 2 was an 83-year-old man with an elevated lesion in the gastric angulus. Biopsy revealed papillary and well-differentiated tubular adenocarcinomas. Pathological diagnosis of the ESD specimen revealed intramucosal gastric cancer without LVI. The entire lesion was occupied by papillary and tubular cancer cells, and had clear vesicles. Pure GAED was diagnosed, because the cells were SALL4 positive. In both cases, resection was curative despite the difference in pathological features.

The SWI/SNF complexes are major regulators of gene expression and their alterations occur in a large array of cancers both of epithelial and mesenchymal lineages. Malignant rhabdoid tumors were the first malignancies linked to deregulation of these complexes with the involvement of SMARCB1 in their development but genetic alterations affect all subunits in other malignancies. In the chest and lung regions, SMARCA4 (BRG1) is the most frequently altered subunit and is involved in the pathogenesis of two subtypes of tumors, including bona fide carcinomas (SMARCA4-deficient non-small cell lung cancers) but also undifferentiated tumors that harbor an undifferentiated phenotype close to those of malignant rhabdoid tumors (SMARCA4-undifferentiated tumors). Although their histogenesis is yet to be fully understood, these tumors are associated with distinct clinical and pathological features even though some overlapping features have been reported in rare cases. SMARCA4 deficiency is easily asserted by immunohistochemistry that show the loss of nuclear expression of the protein in the nuclei of tumor cells. These tumors are commonly associated with high-grade cytological features, rhabdoid cytomorphology, solid architecture and extensive necrosis. The typical immunohistochemical signature of SMARCA4-UT combines co-inactivation of SMARCA2 (BRM) and the overexpression of SOX2 and SALL4. No specific therapeutic strategies have been so far developed for SMARCA4-deficient neoplasms. SMARCB1 subunit is involved in the development of several SMARCB1-deficient sarcomas on top of malignant rhabdoid tumors that may develop in the thorax. Malignant rhabdoid tumors affect mostly children of less than 5y. The differential diagnosis includes epithelioid sarcomas, malignant myoepithelial tumors or myoepithelial carcinomas, extra-skeletal myxoid chondrosarcomas and synovial sarcomas.

Purpose This study aimed to correlate the presence of microlithiasis (ML) in cryptorchidism (CO) patients with the functionality of Sertoli cells and the arrest of gonocyte differentiation. Methods Testicular biopsies were obtained from 21 inguinal CO pediatric patients and were classified in two groups as follows: patients with ML and those without ML. In both groups, the number of Sertoli cells/seminiferous cords and their functionality were determined, considering the concentrations of inhibin B. In addition, the area and the histological alterations of seminiferous epithelium were evaluated. The arrest of gonocyte differentiation was determined by immunoreactivity to SALL4, AP2ɣ, PLAP and POU5F1. Results We found a statistical correlation between the presence of ML with the alterations in the functionality of Sertoli cells without reflecting in the differentiation of the gonocytes. Conclusion The study of this population suggests that the association between CO and ML shows a malfunction of the Sertoli cells without necessarily causing arrest in the differentiation of gonocytes in these patients.

Background MEIS1 (Myeloid ecotropic viral integration site 1) as a homeobox (HOX) transcription factor plays regulatory roles in a variety of cellular processes including development, differentiation, survival, apoptosis and hematopoiesis, as well as stem cell regulation. Few studies have established pluripotency and self-renewal regulatory roles for MEIS1 in human esophageal squamous cell carcinoma (ESCC), and our aim in this study was to evaluate the functional correlation between MEIS1 and the stemness markers in ESCC patients and cell line KYSE-30. Methods Expression pattern of MEIS1 and SALL4 gene expression was analyzed in different pathological features of ESCC patients. shRNA in retroviral vector was used for constantly silencing of MEIS1 mRNA in ESCC line (KYSE-30). Knockdown of MEIS1 gene and the expression pattern of selected stemness markers including SALL4, OCT4, BMI-1, HIWI, NANOG, PLK1, and KLF4 were evaluated using real-time PCR. Results Significant correlations were observed between MEIS1 and stemness marker SALL4 in different early pathological features of ESCC including non-invaded tumors, and the tumors with primary stages of progression. Retroviral knockdown of MEIS1 in KYSE-30 cells caused a noteworthy underexpression of both MEIS1 and major involved markers in stemness state of the cells including SALL4, OCT4, BMI-1, HIWI and KLF4. Conclusions The results highlight the important potential role of MEIS1 in modulating stemness properties of ESCCs and cells KYSE-30. These findings may confirm the linkage between MEIS1 and self-renewal capacity in ESCC and support probable oncogenic role for MEIS1 in the disease.

MicroRNAs are a class of small non-coding RNAs which act as oncogenes or tumor suppressors through targeting specific mRNAs. Colorectal cancer (CRC) is one of the most common malignancies worldwide. MiR-3622a-3p is found to be decreased in colorectal cancer (CRC) by analyzing data from TCGA database and there are few reports about the role of miR-3622a-3p in cancers. Our research aimed to explore the effects of miR-3622a-3p on CRC. MiR-3622a-3p was found to be down-regulated in CRC tissues and cells by qRT-PCR. The effect of miR-3622a-3p on proliferation, apoptosis, cell cycle, migration and invasion of CRC cells were investigated by a serious of biological function assays and the results revealed that miR-3622a-3p could inhibit the malignant biological properties of CRC. We performed dual luciferase assay, RNA immunoprecipitation (RIP) assay and pull-down assay to confirm the interaction between miR-3622a-3p and spalt-like transcription factor 4 (SALL4). Western blot was carried out to determine the effects of miR-3622a-3p and SALL4 on stemness features and EMT. We found that miR-3622a-3p suppressed stemness features and EMT of CRC cells by SALL4 mRNA degradation. MiR-3622a-3p could inhibit CRC cell proliferation and metastasis in vivo with tumor xenograft model and in vivo metastasis model. The CRC organoid model was constructed with fresh CRC tissues and the growth of organoids was suppressed by miR-3622a-3p. Taken together, the results of our study indicate miR-3622a-3p exerts antioncogenic role in CRC by downregulation of SALL4. The research on miR-3622a-3p might provide a new insight into treatment of CRC.

Spermatocyte (spc) formation from spermatogonia (spg) differentiation is the first step of spermatogenesis which produces prodigious spermatozoa for a lifetime. After decades of studies, several factors involved in the functioning of a mouse were discovered both inside and outside spg. Considering the peculiar expression and working pattern of each factor, this review divides the whole conversion of spg to spc into four consecutive development processes with a focus on extracellular cues and downstream transcription network in each one. Potential coordination among Dmrt1, Sohlh1/2 and BMP families mediates Ngn3 upregulation, which marks progenitor spg, with other changes. After that, retinoic acid (RA), as a master regulator, promotes A1 spg formation with its helpers and Sall4. A1-to-B spg transition is under the control of Kitl and impulsive RA signaling together with early and late transcription factors Stra8 and Dmrt6. Finally, RA and its responsive effectors conduct the entry into meiosis. The systematic transcription network from outside to inside still needs research to supplement or settle the controversials in each process. As a step further ahead, this review provides possible drug targets for infertility therapy by cross-linking humans and mouse model.

SUMMARY The 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., In Brief In this paper, Kong et al. elucidate the DNA binding mechanisms of the transcription factor SALL4 and an epigenetic pathway that it regulates. Due to its important role in driving aggressive cancers, better understanding of SALL4 function will lead to strategies to target this protein in cancer., Graphical 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.

Cystic trophoblastic tumor (CTT) is an uncommon trophoblastic proliferation of germ cell tumor origin, mostly reported in post-chemotherapy metastases of testicular germ cell tumors and rarely primary untreated testicular tumors. To date, we are not aware of occurrence in a non-testicular tumor. A 12-year-old boy presented with limb swelling, increased appetite, weight gain, and precocious puberty. Evaluation revealed right frontal lobe mass and elevated α-fetoprotein and β-human chorionic gonadotrophin. After response to neoadjuvant chemotherapy, the tumor was resected. Microscopically, the resection contained predominantly smooth muscle tissue with scattered small foci of glandular teratoma and CTT. Immunohistochemistry (SALL4, glypican 3) revealed no residual yolk sac tumor. Fluorescence in situ hybridization revealed gain of chromosome 12p. The patient has been disease-free for 13 years. This report expands the spectrum of primary central nervous system germ cell tumors with the occurrence of CTT in this site.

Background Yolk sac tumor (YST) is a germ cell neoplasm that arises predominantly in the gonads, but can also derive from somatic neoplasms in extragonadal locations. These latter cases have been documented in several organs, although reports from the urinary tract are limited. To our knowledge, this is the first report of a bladder urothelial carcinoma with a predominant component of YST differentiation. Case presentation We present a unique case of a 76-year-old man with a recurrent urinary bladder tumor, initially interpreted as a high grade urothelial carcinoma with glandular differentiation. In the recurrent tumor, diverse histological patterns were identified, including glandular, hepatoid and sarcomatoid. This tumor showed positivity for AFP, GLP3 and SALL4, and negativity for CK7 and EMA. Fluorescent in situ hybridization study showed a polysomic pattern of chromosome 12. All these findings led to the final diagnosis of a YST derived from urothelial carcinoma. Conclusions YST differentiation should be considered in the differential diagnosis of a high grade urothelial carcinoma, particularly when glandular and other unusual patterns are observed.

Sal-like protein 4 (SALL4) is overexpressed in breast cancer and might contribute to breast cancer progression, but the molecular mechanism remains unknown. Here, we found that within a group of 371 ethnic Chinese breast cancer patients, SALL4 was associated with lower grade (P = .002) and progesterone receptor positivity (P = .004) for overall cases; lower Ki67 (P = .045) and high vimentin (P = .007) for luminal cases. Patients with high SALL4 expression in lymph node metastasis showed a significantly worse survival than those with low expression. Knockout of SALL4 in a triple-negative breast cancer cell line MDA-MB-231-Red-FLuc-GFP led to suppressed ability in proliferation, clonogenic formation, migration, and mammosphere formation in vitro, tumorigenicity and lung colonization in vivo. On the other hand, overexpression of SALL4 enhanced migration and mammosphere formation in vitro and tumorigenicity in vivo. Mechanistically, there was a positive correlation between SALL4 expression and mesenchymal markers including Zinc finger E-box binding homeobox 1 (ZEB1), vimentin, Slug, and Snail in vivo. Chromatin immunoprecipitation experiment indicated that SALL4 can bind to the promoter region of vimentin (-778 to -550 bp). Taken together, we hypothesize that SALL4 promotes tumor progression in breast cancer by inducing the mesenchymal markers like vimentin through directly binding to its promoter. Increased SALL4 level in metastatic lymph node relative to the primary site is an important poor survival marker in breast cancer.

Background Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare subtype of colorectal malignancy with expression of enteroblastic markers (glypican 3, SALL4, AFP); however, the clinicopathological and epidemiological features are not fully elucidated. Aims The aims of this study were to elucidate and establish the molecular and clinicopathological characteristics of CAED. Materials and methods In addition to three cases recently diagnosed as CAED, colorectal carcinoma (CRC) with expression of enteroblastic markers were selected by using immunohistochemistry (IHC) on tissue microarrays of 988 advanced CRC. We employed next-generation sequencing (NGS) and Sanger sequencing for the detection of genetic alterations. IHC for p53 and HER2, HER2-FISH and MSI status was also investigated. Survival analyses for clinicopathological parameters were performed using Kaplan-Meier methods. Results Thirty-nine cases (4.0%) were positive for at least one enteroblastic marker. Histological evaluation of the total of 42 cases revealed that 10 contained tumour cells with clear cytoplasm. Enteroblastic marker-positive cases had aggressive behaviour and poor prognosis. NGS revealed TP53 as the most frequently mutated gene. The rate of HER2-positive cases and MSI-H cases was 9.5% (four of 42) and 12.2% (five of 41), respectively. Among these 42 cases, there were no molecular and clinicopathological differences according to the presence of tumour cells with clear cytoplasm. Conclusions Enteroblastic marker-positive CRC could be grouped together as CAED regardless of clear cell cytoplasm. Using this definition, the frequency of CAED is 4.0% and has a poorer prognosis than that for conventional CRCs. HER2 targeted therapy would be a meaningful treatment for CAED, and CAEDs contain both MSI-H and MSI-stable CRCs, although the MSS phenotype is dominant.

Background Sal-like protein 4 (SALL4), an embryonic stem cell factor, has been reported to play an essential role in embryogenesis and oncogenesis. As yet, however, the expression and role of this transcription factor in head and neck squamous cell carcinoma (HNSCC) has not been established.Methods We assessed SALL4 mRNA expression in a well-characterised dataset of 230 HNSCC samples (test cohort 110 cases and validation cohort 120 cases). We also transfected HNSCC cells (FaDu and UM-SCC-6) with SALL4 siRNA and assessed its effects on proliferation and expression of specific epigenetic factors in order to uncover the role of SALL4 in HNSCC.Results Overexpression of SALL4 was detected in tumour samples of both cohorts. HNSCC cells treated with SALL4 siRNA showed a reduction in growth and a decrease in DNA methyltransferase 3 alpha (DNMT3A) expression. In the patient cohorts, SALL4 overexpression was found to significantly correlate with disease recurrence (p