Sal-like 4 Research Articles

BMI1 fine-tunes gene repression and activation to safeguard undifferentiated spermatogonia fate.

Introduction: Spermatogenesis is sustained by the homeostasis of self-renewal and differentiation of undifferentiated spermatogonia throughout life, which is regulated by transcriptional and posttranscriptional mechanisms. B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), one of spermatogonial stem cell markers, is a member of Polycomb repressive complex 1 (PRC1) and important to spermatogenesis. However, the mechanistic underpinnings of how BMI1 regulates spermatogonia fate remain elusive. Methods: We knocked down BMI1 by siRNA to investigate the role of BMI1 in undifferentiated spermatogonia. Differentially expressed genes were identified by RNA-seq and used for KEGG pathway analysis. We performed ChIP-seq analysis in wild type and BMI1 knockdown cells to explore the underlying molecular mechanisms exerted by BMI1. BMI1-associated alterations in repressive histone modifications were detected via Western blotting and ChIP-seq. Furthermore, we performed mass spectrometry and Co-immunoprecipitation assays to investigate BMI1 co-factors. Finally, we demonstrated the genomic regions occupied by both BMI1 and its co-factor. Results: BMI1 is required for undifferentiated spermatogonia maintenance by both repressing and activating target genes. BMI1 preserves PI3K-Akt signaling pathway for spermatogonia proliferation. Decrease of BMI1 affects the deposition of repressive histone modifications H2AK119ub1 and H3K27me3. BMI also positively regulates H3K27ac deposited genes which are associated with proliferation. Moreover, we demonstrate that BMI1 interacts with Sal-like 4 (SALL4), the transcription factor critical for spermatogonia function, to co-regulate gene expression. Discussion: Overall, our study reveals that BMI1 safeguards undifferentiated spermatogonia fate through multi-functional roles in regulating gene expression programs of undifferentiated spermatogonia.

ALDH1 and SALL4 Expression in Cell Block Samples from Patients with Lung Adenocarcinoma and Malignant Pleural Effusion.

Malignant pleural effusion (MPE) can accompany advanced lung adenocarcinoma. Recent studies suggest that MPE could contain a heterogeneous subpopulation of cells with stem-like properties, such as tumorigenicity and self-renewal, indicating that they could be the source of metastasis. Although previous studies analyzed the correlation between cancer stem cell (CSC) marker expression and clinical outcomes using lung cancer tissues, investigations regarding the association of MPE with CSC marker expression are limited. We performed immunohistochemistry to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and Sal-like 4 (SALL4) in 46 cell block samples of MPE from patients with lung adenocarcinoma. ALDH1-positive and SALL4-positive cancer cells in MPE were detected in 30 (65.2%) and 21 samples (45.7%), respectively. Cluster formation was detected in 26 samples (56.5%). The number of clusters was significantly higher in ALDH1-positive/SALL4-negative samples. SALL4 expression was inversely correlated with the cluster ratio (r = −0.356) and positively associated with the Ki-67 index (r = 0.326), suggesting that MPE cells with high SALL4 expression comprised the proliferative subpopulation. In conclusion, we demonstrated that MPE contains an ALDH1-positive/SALL4-negative subpopulation exhibiting cluster formation and a SALL4-positive proliferative subpopulation.

Effect of SALL4 on the Proliferation, Invasion and Apoptosis of Breast Cancer Cells.

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.

Upregulation of SNHG12 accelerates cell proliferation, migration, invasion and restrain cell apoptosis in breast cancer by enhancing regulating SALL4 expression via sponging miR-15a-5p.

Breast cancer (BC) is malignant cancer that threatens the health of millions of females worldwide. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) has been identified as an oncogene in multiple cancers. However, the regulatory role of SNHG12 in BC cell progression is still obscured. The levels of SNHG12, miR-15a-5p, and Sal-like 4 (SALL4) in BC tumor tissues and cells were measured by qRT-PCR. Cell viability, apoptosis, migration, and invasion were examined by CCK8, flow cytometry, and transwell assay, respectively. The interaction between miR-15a-5p and SNHG12 or SALL4 was evaluated by dual-luciferase reporter assay. Protein expression of SALL4 was analyzed by western blot. Xenograft mice were established by subcutaneously injecting BC cells stably transfected with sh-SNHG12 and sh-NC. SNHG12 and SALL4 expressions were upregulated whereas miR-15a-5p was downregulated in BC tumors compared with normal tissues. Besides, miR-15a-5p was correlated with SNHG12 and SALL4 inversely as calculated by Pearson's correlation coefficient. More importantly, SNHG12 knockdown attenuated BC tumor growth in vitro and in vivo. Subsequently, dual-luciferase reporter assay confirmed the interaction between miR-15a-5p and SNHG12 or SALL4. The rescue experiments revealed that miR-15a-5p inhibitor restored SNHG12 silencing induced inhibition on BC cell proliferation, migration, invasion, and promotion of apoptosis. Additionally, SNHG12 was found to accelerate BC cell progression by absorbing miR-15a-5p to enhance SALL4 expression. SNHG12 promotes cell proliferation, migration, and invasion but suppresses apoptosis in BC by upregulating SALL4 expression via sponging miR-15a-5p, representing potential targets for the development of novel diagnosis and treatment methods.

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable.

Sal-like 4 protein levels in breast cancer cells are post-translationally down-regulated by tripartite motif–containing 21

Sal-like 4 (SALL4) is a transcription factor that enhances proliferation and migration in breast cancer cells. SALL4 expression therefore has the potential to promote cancer malignancy. However, the regulatory mechanisms involved in SALL4 protein expression have not been thoroughly elucidated. In this study, we observed that treating MCF-7 and SUM159 breast cancer cell lines with a proteasome inhibitor increases SALL4 protein levels, suggesting that SALL4 is degraded by the ubiquitin-proteasome system. Using immunoprecipitation to uncover SALL4-binding proteins, we identified an E3 ubiquitin-protein ligase, tripartite motif-containing 21 (TRIM21). Using an EGFP reporter probe of the major SALL4 isoform SALL4B, we observed that shRNA-mediated knockdown of TRIM21 increases cellular SALL4B levels. Immunostaining experiments revealed that TRIM21 localizes to the nucleus, and a K64R substitution in the nuclear localization motif in SALL4B increased SALL4B levels in the cytoplasm. These results suggested that TRIM21 is involved in nuclear SALL4 degradation. To identify the amino acid residue that is targeted by TRIM21, we fragmented the SALL4B sequence, fused it to EGFP, and identified Lys-190 in SALL4B as TRIM21's target residue. Amino acid sequence alignments of SALL family members indicated that the region around SALL4 Lys-190 is conserved in both SALL1 and SALL3. Because SALL1 and SALL4 have similar functions, we constructed a SALL1-EGFP probe and found that the TRIM21 knockdown increases SALL1 levels, indicating that TRIM21 degrades both SALL1 and SALL4. Our findings extend our understanding of SALL4 and SALL1 regulation and may contribute to the development of SALL4-targeting therapies.

The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells

During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration.

MicroRNA-33b inhibits cell proliferation in hepatocellular carcinoma via targeting SALL4

To investigate the expression of miR-33b in hepatocellular carcinoma (HCC) and to explore regulatory mechanism of miR-33b for cell proliferation of HCC. HCC tissues and adjacent non-tumor tissues were collected for this study (n=32 for each). Real-time PCR and Western blot were conducted to examine the mRNA and protein expression, respectively. MTT assay was used to detect the cell proliferation. Luciferase reporter gene assay was performed to verify the target relationship between miR-33b and Sal-like 4 (SALL4). MiR-33b was significantly downregulated in HCC tissues compared with adjacent non-tumor tissues. Overexpression of miR-33b decreased the proliferation of HCC LH86 cells. SALL4 was identified as a target gene of miR-33b, and its protein expression was negatively regulated by miR-33b. Overexpression of SALL4 reversed the suppressive effect of miR-33b on LH86 cell proliferation. SALL4 was significantly upregulated in HCC tissues compared with adjacent non-tumor tissues. The miR-33b suppresses HCC cell proliferation through down-regulation of SALL4.

Expression of sal-like 4 in primary hepatocellular carcinoma and its association with epithelial-mesenchymal transition

Objective: To investigate the expression of sal-like 4 (SALL4) in tissues of primary hepatocellular carcinoma (HCC) and its association with epithelial-mesenchymal transition (EMT) and clinicopathological characteristics. Methods: Immunohistochemistry and nucleic acid in situ hybridization were used to measure the mRNA expression of SALL4, epithelial cadherin (E-cadherin), vimentin, and Snail in 72 HCC samples, 2 fetal liver samples, and 2 normal adult liver tissue samples. Results: Strong expression of SALL4 was observed in hepatoblasts in fetal liver, but SALL4 expression was not observed in primitive hematopoietic cells and normal adult hepatocytes or biliary epithelial cells. In the HCC samples, the positive rate of SALL4 was 47.2% (34/72), showing focal positive nuclear staining. The HCC patients with microscopic microvascular tumor thrombus and portal vein tumor thrombus, a serum alpha-fetoprotein level of≥ 350 ng/ml, International Union Against Cancer (UICC) stage III+IV, and an age of < 46 years showed higher positive expression of SALL4 than those with no microscopic microvascular tumor thrombus or portal vein tumor thrombus, a serum alpha-fetoprotein level of < 350 ng/ml, UICC stage I+II, and an age of≥46 years. The HCC patients with positive SALL4 showed lower postoperative disease-free survival rate and overall survival rate than those with negative SALL4 (P < 0.05). Sixty percent of the patients with microvascular tumor thrombus (21/35) showed positive expression of SALL4. The positive rate of SALL4 was negatively correlated with E-cadherin (r = -0.434, P < 0.01), but positively correlated with vimentin and Snail (vimentin: r = 0.516, P < 0.01; Snail: r = 0.571, P < 0.01). Conclusion: In patients with primary HCC, the expression of SALL4 greatly affects EMT, which helps with the research on invasion and metastasis of liver cancer and prognostic evaluation.

Expression and clinical significance of sal-like 4 in thyroid carcinoma

Objective To investigate the expression of sal-like 4 (SALL4) gene in thyroid carcinoma and analyze its clinical significance. Methods The mRNA and protein expression of SALL4 was separately detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting in 60 surgically resected specimens of thyroid carcinoma and their relative paracancerous tissue specimens. The correlation between the expression of SALL4 mRNA and protein and clinicopathologic parameters of thyroid carcinoma was analyzed with SPSS 17.0. Results The expression of SALL4 mRNA (85%) and protein (80%) was significantly higher in thyroid carcinoma tissues than in the adjacent tissues. SALL4 mRNA and protein expression in thyroid cancer tissues was correlated with TNM stage and lymph node metastasis (P<0.05), but not with gender, age, tumor size and differentiation of tissue pathology. The expression of SALL4 protein was mostly accompanied by SALL4 mRNA (P<0.05). Conclusion The expression of SALL4 was increased in thyroid cancer, and may be involved in progression and metastasis of thyroid cancer. Key words: Thyroid cancer; Sal-like 4; Western blotting

Knockdown of SALL4 inhibits the proliferation and reverses the resistance of MCF-7/ADR cells to doxorubicin hydrochloride.

BackgroundBreast cancer is the most frequent malignancy in women and drug resistance is the major obstacle for its successful chemotherapy. In the present study, we analyzed the involvement of an oncofetal gene, sal-like 4 (SALL4), in the tumor proliferation and drug resistance of human breast cancer.ResultsOur study showed that SALL4 was up-regulated in the drug resistant breast cancer cell line, MCF-7/ADR, compared to the other five cell lines. We established the lentiviral system expressing short hairpin RNA to knockdown SALL4 in MCF-7/ADR cells. Down-regulation of SALL4 inhibited the proliferation of MCF-7/ADR cells and induced the G1 phase arrest in cell cycle, accompanied by an obvious reduction of the expression of cyclinD1 and CDK4. Besides, down-regulating SALL4 can re-sensitize MCF-7/ADR to doxorubicin hydrochloride (ADMh) and had potent synergy with ADMh in MCF-7/ADR cells. Depletion of SALL4 led to a decrease in IC50 for ADMh and an inhibitory effect on the ability to form colonies in MCF-7/ADR cells. With SALL4 knockdown, ADMh accumulation rate of MCF-7/ADR cells was increased, while the expression of BCRP and c-myc was significantly decreased. Furthermore, silencing SALL4 also suppressed the growth of the xenograft tumors and reversed their resistance to ADMh in vivo.ConclusionSALL4 knockdown inhibits the growth of the drug resistant breast cancer due to cell cycle arrest and reverses tumor chemo-resistance through down-regulating the membrane transporter, BCPR. Thus, SALL4 has potential as a novel target for the treatment of breast cancer.

SALL4 and its clinical application value in non-germ cell tumors

Sal-like 4 (SALL4) plays an important role in promoting the cellular proliferation and maintaining the pluripotency of embryonic stem cells and tumor cells. In fully differentiated cells, the expression of SALL4 is silenced or down-regulated. However, the expression of SALL4 is found to be restored or up-regulated in a variety of non-germ cell tumors. Besides, the expression of SALL4 is often associated with disease progression, treatment effect and prognosis. Therefore, examining the expression level of SALL4 will be of great importance in the diagnosis of disease and monitoring the disease progression for non-germ cell tumors. Key words: Neoplasms; Diagnosis; Tumor markers, biological; Sal-like 4

Expression and clinical significance of SALL4 and β-catenin in colorectal cancer.

Previous studies on the expression of Sal-like 4 (SALL4), a zinc finger transcription factor, had conflicting results in colorectal cancer (CRC). The main aim of this study was to investigate the expression of SALL4 and its relationship with β-catenin in CRC. Immunohistochemistry was performed to examine the expression of SALL4 and β-catenin in a cohort of 149 patients with CRC, 12 with atypical hyperplasia, 25 with benign tumor and 38 with normal tissue. Expression patterns of SALL4 and β-catenin and correlation with clinicopathological features were investigated. The relationship between SALL4 and β-catenin was examined by immunofluorescence and co-immunoprecipitation using CRC cell lines, SW480, SW620, HCT116 and HT29. Immunohistochemical analysis revealed significantly lower expression of SALL4 in CRC (46.3 %) than atypical hyperplasia (68.0 %, p < 0.05) and normal tissue (78.9 %, p < 0.01). Well-differentiated CRC seemed to express more SALL4 (47.6 %) than moderately (45.8 %) and poorly-differentiated cancers (18.8 %) (p < 0.05). However, SALL4 expression positively correlated with lymph node metastasis and tumor-node-metastasis (TNM) and Dukes stages (all p < 0.05) suggesting a new mechanism involved in the function of SALL4 in CRC. β-catenin was expressed significantly higher in CRC (69.1 %) than normal tissue (21.1 %, p < 0.01), and positively correlated with CA19-9 level in serum (p < 0.05). SALL4 and β-catenin were positively correlated in CRC (Spearman correlation coefficient R = 0.536, p < 0.01). The group of co-expression of the two molecules showed advanced lymph node metastasis, TNM stage and Dukes stage (all p < 0.05). Double-labeling immunofluorescence and co-immunoprecipitation indicated that SALL4 and β-catenin co-localized in the nucleus and cytoplasm and interacted. Taken together, our results revealed that SALL4 and β-catenin were positively correlated in CRC. In CRC cells, SALL4 and β-catenin co-localized and interacted. The function of SALL4 in promoting lymph node metastasis and advanced clinical stage might partly be due to the interaction with β-catenin.

The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout adulthood through balanced self-renewal and differentiation, yet the regulatory logic of these fate decisions is poorly understood. The transcription factors Sal-like 4 (SALL4) and promyelocytic leukemia zinc finger (PLZF; also known as ZBTB16) are known to be required for normal SSC function, but their targets are largely unknown. ChIP-seq in mouse THY1(+) spermatogonia identified 4176 PLZF-bound and 2696 SALL4-bound genes, including 1149 and 515 that were unique to each factor, respectively, and 1295 that were bound by both factors. PLZF and SALL4 preferentially bound gene promoters and introns, respectively. Motif analyses identified putative PLZF and SALL4 binding sequences, but rarely both at shared sites, indicating significant non-autonomous binding in any given cell. Indeed, the majority of PLZF/SALL4 shared sites contained only PLZF motifs. SALL4 also bound gene introns at sites containing motifs for the differentiation factor DMRT1. Moreover, mRNA levels for both unique and shared target genes involved in both SSC self-renewal and differentiation were suppressed following SALL4 or PLZF knockdown. Together, these data reveal the full profile of PLZF and SALL4 regulatory targets in undifferentiated spermatogonia, including SSCs, which will help elucidate mechanisms controlling the earliest cell fate decisions in spermatogenesis.

Effects of siRNA-mediated silencing of Sal-like 4 expression on proliferation and apoptosis of prostate cancer C4-2 cells.

The aim of this study was to evaluate the effects of small interfering RNA (siRNA)-inhibited expression of the Sal-like 4 (SALL4) gene on the proliferation, colony formation, and apoptosis of prostate cancer C4-2 cells. C4-2 cells were cultured and divided into a si-SALL4 group, a negative control siRNA group, and a blank control group. SALL4 mRNA levels and protein expression were detected by real-time polymerase chain reaction and western blot, respectively. Changes in the cell proliferation and colony formation capacities were observed by using the MTS colorimetric method and colony formation assay, respectively. The influence of SALL4 on apoptosis was assessed with flow cytometry, and the expression of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2) and bcl-like-protein 4 (Bax) were detected by western blot. The si-SALL4 group had significantly lower mRNA and protein levels of SALL4 as well as decreased proliferation and colony formation capacities than the negative control group (P < 0.05). There were significantly more apoptotic cells in the si-SALL4 group compared to the negative control (P < 0.05), and the expression of Bcl-2 and Bax decreased and increased, respectively, after treatment with si-SALL4. Silencing SALL4 expression by using siRNA technology inhibited the proliferation and colony formation of C4-2 cells, and promoted apoptosis likely mediated by Bcl-2 and Bax expression. These results provide experimental basis for further elucidating the role of SALL4 in prostate cancer cells.

High cytoplasmic expression of SALL4 predicts a malignant phenotype and poor prognosis of breast invasive ductal carcinoma

Sal-like 4(SALL4) is significant for maintaining self-renewal and pluripotency in embryonic stem cells, cancer cells and perhaps even cancer stem cells. The expression of SALL4 has been recorded in various kinds of cancers and is deemed to have aclinical value for diagnosis. However, little information on SALL4 expression has been illustrated in breast cancer. In this study, the expression of SALL4 was scrutinized by immunohistochemical analysis in breast invasive ductal carcinoma in alarge cohort of 160 patients. High cytoplasmic expression of SALL4 was detected in breast cancer tissues compared with normal adjacent tissues. High SALL4 expression was associated with advanced tumor invasion (p = 0.019), lymph node stage (p = 0.027), ER (p = 0.030), PR (p = 0.037), HER2 (p = 0.019) and TNBC (p = 0.007) in overall patients. Interestingly, in Kaplan-Meier analysis, breast cancer patients with high expression of SALL4 demonstrated aworse OS. Both univariate and multivariate analysis illustrated that examination of SALL4 was of great prognostic value in OS. Thus, our data showed that high cytoplasmic expression of SALL4 was considered to be an independent prognostic indicator for breast invasive ductal carcinoma.

SALL4: engine of cell stemness.

The spalt (sal) family is a class of evolutionarily conserved genes originally identified in Drosophila as homeotic genes required for embryonic development. In vertebrates, the expression of sal-like 4 (SALL4) is specifically enriched in both embryonic and adult stem/stem-like cells. SALL4 is a master regulator that contributes to cell stemness in biological development and tumor growth. Thus, Sall4 has emerged as a target for gene therapy. In addition, numerous mutations affecting the Sall4 gene have been discovered and clinically linked to a series of congenital abnormalities, such as Duane/Duane-related syndromes, ventricular septal defect and premature ovarian failure. This review delineates the underlying mechanisms of key functions of SALL4 and its use as a target for gene therapy. Finally, I summarize and discuss advances made on the application of Sall4 and its functions in diagnostics and treatments for human diseases.

Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal-like 4 (SALL4) as a dispersion factor in basal-like breast cancer. Our shRNA-mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal-like breast cancer.

RNA-binding protein LIN28 is a marker for testicular germ cell tumors

LIN28 is an RNA-binding protein involved in maintaining the pluripotency of embryonic stem cells. Using formalin-fixed, paraffin-embedded tissue blocks, we performed immunohistochemical staining of LIN28 in 103 primary and 81 metastatic testicular germ cell tumors (54 intratubular germ cell neoplasias, unclassified type; 49 primary and 20 metastatic classic seminomas; 35 primary and 24 metastatic embryonal carcinomas; 35 primary and 15 metastatic yolk sac tumors; 23 primary and 12 metastatic teratomas; 6 primary and 10 metastatic choriocarcinomas; and 5 spermatocytic seminomas). The percentage of tumor cell stained was scored as 0 (0%), 1+ (≤30%), 2+ (31%-60%), 3+ (61%-90%), and 4+ (>90%). We stained LIN28 in 327 non-germ cell tumors to determine its specificity. We also compared LIN28 with SALL4 (Sal-like 4) and OCT4 (octamer-binding transcription factor 4) in all germ cell tumors. The staining was cytoplasmic for LIN28 and nuclear for SALL4 and OCT4. Strong 4+ LIN28 staining was seen in all 54 intratubular germ cell neoplasias, 59 embryonal carcinomas, and 50 yolk sac tumors. Positive LIN28 staining was seen in all 69 classic seminomas (1+ in 3, 3+ in 3, and 4+ in 63) (63, strong). Variable staining of LIN28 was seen in 10 of 35 teratomas (1+ to 3+, weak to strong intensity), 12 of 16 choriocarcinomas (1+ to 4+, weak to strong intensity), and 1 of 5 spermatocytic seminomas (2+, weak). Only 10 of 327 non-germ cell tumors showed 1+ weak LIN28 staining. Therefore, LIN28 is a highly sensitive marker for testicular intratubular germ cell neoplasias, classic seminomas, embryonal carcinomas, and yolk sac tumors with relatively high specificity. LIN28 can be used as a diagnostic marker for these tumors and has demonstrated a similar level of diagnostic utility as SALL4 (except for a few classic seminomas), although it does not show an advantage over SALL4. The major advantage of LIN28 over OCT4 is in diagnosing yolk sac tumors (yolk sac tumors negative for OCT4).

Genetic variation of SAL-Like 4 (SALL4) in ventricular septal defect

Ventricular septal defect (VSD) accounts for about half of congenital heart disease (CHD). SAL-Like 4 (SALL4) gene mutations have been identified to be the cause of Okihiro syndrome which is characterized by association limb and multiple other organ developmental defects including heart defect. We screened SALL4 gene coding regions for mutations using DNA sequencing approach in 300 nonsyndromic VSD patients and 250 with no reported cardiac phenotype controls. We discovered two novel variants: c.586C>T (p.Arg196Trp) and c.2389A>T (p.Ser797Cys) in 300 nonsyndromic VSD patients. The two non-synonymous variations were located in the SALL4 evolutionarily conserved residues and the amino acid change may affect the function of SALL4. Our finding is the first to suggest that SALL4 may be a potential candidate gene of ventricular septal defect (VSD).