Two Cases of Gastric Adenocarcinoma with Enteroblastic Differentiation which Demonstrated Rapid Progressive Courses.

We herein report two extremely rare cases of gastric adenocarcinoma with enteroblastic differentiation (GAED) that underscore the aggressive nature of GAED. Case 1: ESD was scheduled for early-stage gastric cancer, however, the tumor increased in size drastically and the morphology changed to type "0-I + IIc" in one month. Surgery was performed and the patient was diagnosed with GAED. Case 2: ESD was performed for early-stage gastric cancer, and the pathological findings revealed GAED. The horizontal margin was positive for clear cells in the muscularis mucosa. Additional surgery was performed; however, recurrence occurred one year later. Therefore, the treatment strategies should be carefully considered for GAED.

[Retracted] MicroRNA-33b suppresses the proliferation and metastasis of hepatocellular carcinoma cells through the inhibition of Sal-like protein 4 expression.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the Transwell cell migration data shown in Figs. 2D and 4C were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive any reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 38: 1587­1595, 2016; DOI: 10.3892/ijmm.2016.2754].

[Retracted] Methylation­induced downregulation and tumor-suppressive role of microRNA­98 in glioma through targeting Sal­like protein 4.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the cell Transwell assay data in the article (featured in Figs. 4D and 6D) were strikingly similar to data appearing in different form in other articles by different authors at different research institutions, which were already under consideration for publication or had already been published elsewhere at the time of the present article's submission. Owing to the fact that the contentious data in the above article had already appeared in different form in other articles prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 2651-2659, 2018; DOI: 10.3892/ijmm.2018.3464].

Comparison of immunohistochemical profiles of ovarian germ cells in dysgerminomas of a captive maned wolf and domestic dogs.

We characterized the immunohistochemical expression profiles of dysgerminomas from a 16-y-old maned wolf and 13 domestic dogs using the following biomarkers: Sal-like protein 4 (SALL4), octamer-binding transcription factor 3/4 (OCT3/4), placental alkaline phosphatase (PLAP), c-kit, and vimentin. The maned wolf had nonspecific and long-standing clinical signs of lethargy, anorexia, and weight loss, and was euthanized because of poor prognosis. At autopsy, the left ovary was effaced by a 12 × 8 × 6 cm mass, comprised of anaplastic cells with a mitotic count of 20 mitoses in 10 high power fields. Dysgerminomas from 7 of 13 domestic dogs had nuclear expression of SALL4. Dysgerminomas from the maned wolf and 2 domestic dogs had both nuclear and cytoplasmic expression of SALL4. Cytoplasmic expression of PLAP and OCT3/4 was present in dysgerminomas from the maned wolf and 3 (PLAP) or 4 (OCT3/4) domestic dogs. All dysgerminomas expressed vimentin. Membranous c-kit expression was rare in the dysgerminoma from the maned wolf, and variable in dysgerminomas from 4 domestic dogs. A dysgerminoma from a domestic dog had cytoplasmic expression of c-kit. SALL4 is a useful marker to confirm germ cell origin of dysgerminoma in canids.

Mixed germ cell tumor infiltrating the pineal gland without elevated tumor markers: illustrative case.

BACKGROUND: Tumors in the pineal region consist of various histological types, and correct diagnosis from biopsy specimens is sometimes difficult. The authors report the case of a patient with a mixed germ cell tumor infiltrating into the pineal gland despite showing no elevation of tumor markers. OBSERVATIONS: An 18-year-old man complained of headache and nausea and showed disturbance of consciousness. Magnetic resonance imaging showed hydrocephalus associated with a cystic pineal tumor. The patient underwent tumor biopsy followed by endoscopic third ventriculostomy for hydrocephalus in a local hospital. A pineocytoma was diagnosed, and the patient was referred to the authors' hospital for treatment. Concentrations of placental alkaline phosphatase, alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin in cerebrospinal fluid were not elevated. However, the authors' review of the tumor specimen revealed some immature cells infiltrating the pineal gland. These cells were positive for AFP, Sal-like protein 4, and octamer-binding transcription factor 3/4; and the diagnosis was changed to mixed germ cell tumor. Chemoradiotherapy was initiated, followed by surgical removal of the residual tumor. LESSONS: Careful examination of all tumor specimens and immunohistochemical analyses are important for accurate diagnosis of pineal tumors.

Knockdown of SALL4 inhibits the proliferation, migration, and invasion of human lung cancer cells in vivo and in vitro.

BACKGROUND: This study aimed to investigate the SALL4 expression in lung cancer, determine if SALL4 regulates the biological functions of lung cancer cells at the cellular level, and clarify the possible mechanisms involved. METHODS: Immunohistochemistry was used to detect the SALL4 expression messenger RNA (mRNA) in 62 cases of lung cancer tissue microarray. The correlation of SALL4 with the clinical pathological parameters and overall life cycle of patients and the impact of disease-free life cycle was analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the SALL4 expression in lung cancer cell lines and nude mouse models. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, colony-forming assay, and flow cytometry were used to detect the effects of interference with SALL4 expression on lung cancer cell proliferation and transplant tumor models; the effect of interference with SALL4 expression on the growth of transplanted tumors in vivo was also examined. RESULTS: SALL4 was highly expressed in lung cancer tissues and cell lines and was closely related to the patient's TNM stage and lymph node metastasis. Compared to patients with a high SALL4 expression, those with a lower SALL4 expression had a longer overall and disease-free survival. The expression of SALL4 is an independent risk factor for the prognosis of lung cancer patients. Interference with SALL4 expression can significantly inhibit cell proliferation and clonal formation. Interfering with the expression of SALL4 can arrest the cells in the G0/G1 phase by inhibiting the expression of the cell cycle-related proteins, cyclin B, cyclin E, and cyclin D1. Furthermore, wound-healing and Transwell assays showed that interference with SALL4 expression could significantly inhibit the migration and invasion of lung cancer cells, while experiments in nude mice showed that interference with SALL4 expression could significantly inhibit the size and weight of transplanted tumors. CONCLUSIONS: SALL4 was highly expressed in lung cancer cell lines. Interference with the expression of SALL4 can effectively inhibit the proliferation, migration and invasion of lung cancer cells, promote cell cycle arrest, and play the function of tumor suppressor genes. SALL4 may be a new target for the diagnosis and treatment of lung cancer.

miR-188-5p emerges as an oncomiRNA to promote gastric cancer cell proliferation and migration via upregulation of SALL4.

MicroRNAs (miRNAs) play pivotal roles in modulating key biological processes in gastric cancer (GC). As a newly identified miRNA, the function and potential mechanism of miR-188-5p in GC has not been thoroughly elucidated. Here, quantitative real-time polymerase chain reaction detection showed abnormally higher expression of miR-188-5p in GC cells and tissues. Gain-of-function analysis in vitro showed that miR-188-5p promoted GC cell proliferation and migration, while loss-of-function studies showed the reverse. Targetscan has predicted that phosphatase and tensin homolog (PTEN) was a potential target gene of miR-188-5p. miR-188-5p suppressed PTEN messenger RNA and protein expression and activated downstream AKT/mTOR signaling in GC cells, but luciferase reporter analysis showed that PTEN was not regulated by miR-188-5p via the 3' untranslated region. Furthermore, we observed that miR-188-5p overexpression promoted Sal-like protein 4 (SALL4) protein expression, cellular nuclear translocation, and transcription. Knockdown of SALL4 eliminated the effect of miR-188-5p in GC cells as well as suppression of PTEN. Taken together, our results demonstrate that miR-188-5p promotes GC cell proliferation and migration while suppressing tumor suppressor gene PTEN expression via transcriptional upregulation of oncogene SALL4. We conclude that miR-188-5p acts as an oncomiRNA in GC and may be a promising therapeutic target for GC.

The Prognostic Value of Cytokeratin and Sal-Like Protein 4 Expression in Hepatocellular Carcinoma and Intra-Hepatic Cholangiocarcinoma in Taiwan.

Background: We previously reported that modulation of cytokeratin18 induces pleomorphism of liver cells, higher cell motility, and higher drug sensitivity to sorafenib treatment of hepatoma cells. These relationships were established by in vitro experiments. The aim of this study was to determine the in vivo association between cytokeratin expression and tumor behavior, as well as cancer stem cells of hepatocellular carcinoma and intra-hepatic cholangiocarcinoma in Taiwan. Methods: Cytokeratins and sal-like protein 4 expression was determined in 83 hepatocellular carcinoma and 30 intra-hepatic cholangiocarcinoma specimens by immunohistochemistry. The relationship between cytokeratins and sal-like protein 4 expression with hepatitis virus infection, clinicopathologic factors, and survival was analyzed. Further, the correlation among cytokeratins and sal-like protein 4 expression was studied. Results: In addition to cytokeratin8/18, the expression of cytokeratin7/19 and sal-like protein 4 was noted in hepatocellular carcinoma; however, only cytokeratin19 expression had a significant correlation with poor overall survival and poor disease-free survival. The expression of cytokeratins and sal-like protein 4 was not correlated with hepatitis virus infection. The expression of cytokeratin19, but not 7, 8, and 18, was correlated with sal-like protein 4 expression in hepatocellular carcinoma. Cytokeratin7 expression was decreased and the sal-like protein 4 expression was absent in all 30 intra-hepatic cholangiocarcinoma cases. The expression of cytokeratins had not statistically significant correlation with overall and disease-free survival in patients with intra-hepatic cholangiocarcinoma. Conclusions: The expression of cytokeratin19 was associated with sal-like protein 4 expression, as well as poor overall and disease-free survival in hepatocellular carcinoma patients in Taiwan.

Knockdown of SALL4 overcomes cisplatin-resistance through AKT/mTOR signaling in lung cancer cells.

Drug resistance is an important factor for the poor prognosis of non-small cell lung cancer (NSCLC). Sal-like protein 4 (Sall4) is a stem cell marker, and plays a role in maintaining self-renewal. Previous studies have demonstrated that Sall4 may be a candidate for use as support in the diagnosis of lung cancer, and may also represent a therapeutic target. However, the role of Sall4 on drug resistance of lung cancer cells and the mechanism by which Sall4 regulates the sensitivity of lung cancer cells to cisplatin (DDP) remains unknown. In this study, we aim to investigate whether knockdown of Sall4 by siRNA can enhance the apoptosis induced by cisplatin in lung cancer cells. We here reported that the expression of Sall4 was dramatically upregulated in cisplatin-resistant A549 cells compared with the parental cells. Knockdown of Sall4 by siRNA in cisplatin-resistant A549 cells reduced the IC50 compared with the parental cells. In addition, knockdown of Sall4 significantly inhibited cell proliferation, induced apoptosis and invasion cisplatin-resistant A549 cells through AKT/mTOR signaling. Our findings demonstrate that Sall4 is an essential regulator in cisplatin-induced apoptosis, and knockdown of Sall4 may restore cisplatin sensitivity in acquired resistant cells. Thus, our study provides an effective therapeutic strategy for NSCLC treatment.

MicroRNA-219 exerts a tumor suppressive role in glioma via targeting Sal-like protein 4.

MicroRNAs (miRs) serve important roles in the development and progression of various human cancer types, including glioma. Recently, miR-219 has been suggested to function as a tumor suppressor in glioma; however, the underlying mechanism remains largely unknown. The aim of this study was to investigate the regulatory mechanism of miR-219 in the malignant phenotypes of glioma cells. Quantitative polymerase chain reaction (qPCR) and western blotting were conducted to examine the mRNA and protein expression. An MTT assay, wound healing assay and Transwell assay were used to study cell proliferation, migration and invasion. The qPCR data indicated that the expression of miR-219 was significantly decreased in glioma tissues compared with normal brain tissues. In addition, a low expression of miR-219 was identified to be associated with an advanced pathological grade. In vitro experiments demonstrated that miR-219 was also downregulated in several common glioma cell lines, including A172, U87, U251 and U373, when compared with that in normal astrocytes. Ectopic expression of miR-219 caused a significant decrease in U87 cell proliferation, migration and invasion. Luciferase reporter assay data indicated that Sal-like protein 4 (SALL4) was a direct target gene of miR-219, while the protein expression of SALL4 was negatively regulated by miR-219 in U87 cells. Furthermore, SALL4 was significantly upregulated in glioma tissues and cell lines, and upregulation of SALL4 was associated with a higher pathological grade. Furthermore, overexpression of SALL4 significantly attenuated the suppressive effects of miR-219 on U87 cell proliferation, migration and invasion, suggesting that miR-219 serves a suppressive role in glioma growth and metastasis via targeting SALL4. Therefore, the present study highlighted the clinical significance of the miR-219/SALL4 axis in glioma.

Analysis of putative biomarkers of undifferentiated spermatogonia in dog testis.

Spermatogenesis begins after puberty and continues throughout a male's life, and is regulated by spermatogonial stem cells in the seminiferous tubules. Markers of male germ cells, including undifferentiated spermatogonia to fully developed spermatozoa have been identified in rodents, but not in dogs. In this study, to characterize the markers of undifferentiated spermatogonia, histological and immunohistochemical analyses were performed on pre-pubertal (1-month-old), early pubertal (4-month-old), and post-pubertal (7-month-old) dog testes. Expression of chemokine receptor 4 (CXCR4), insulin-like growth factor binding protein 3 (IGFBP3), LIN28, and Sal-like protein 4 (SALL4) genes was confirmed by immunohistochemical analysis. In pre-pubertal and early pubertal dog testes, CXCR4, IGFBP4, and LIN28 genes were expressed in undifferentiated spermatogonia, whereas the SALL4 gene was not expressed in the pre-pubertal stage. In adult dog testes, CXCR4 and IGFBP3 gene expression was detected in undifferentiated spermatogonia and co-localized with protein gene product 9.5 (PGP9.5) near the basement membrane of the seminiferous tubules. The LIN28 and SALL4 genes were expressed in synaptonemal complex protein 3-positive spermatocytes. The CXCR4 and IGFBP3 gene expression is conserved among other species, while LIN28 and SALL4 gene expression varies. Based on results of the present study, it is suggested that undifferentiated spermatogonia markers detected in other species are conserved in dogs. These results may facilitate further studies of the cellular mechanisms of spermatogenesis in dogs.

Stage-specific expression of Sal-like protein 4 in boar testicular germ cells.

Spermatogenesis, the complex process of sperm cell development including mitotic cell division and meiosis, relies on spermatogonial stem cells (SSCs). While markers for developing germ cells have been well investigated in mice, developmental stage-specific markers of germ cells in domestic animals have not been identified. Sal-like protein 4 (SALL4) is known as a putative marker for undifferentiated spermatogonia in rodents; however, its expression in domestic animals has not been investigated. The objective of this study was to characterize the expression of SALL4 in the developmental stages of boar testes and SSCs. Interestingly, all SALL4-expressing cells responded positively to PGP9.5, which is known as a spermatogonia marker in boar testes, while some PGP9.5-positive cells did not express SALL4 in pre-pubertal boar testes. At this stage, the expression of SALL4 was observed in GFRα1-positive cells, and its expression was maintained in cultured pSSCs in vitro, suggesting that SALL4 is a marker of early-stage boar spermatogonia that express GFRα1 in pre-pubertal testes. Additionally, SALL4 expression was observed in c-Kit-positive but not in PGP9.5- or SCP3-positive cells in post-pubertal testes. In conclusion, SALL4 is expressed in early undifferentiated spermatogonia in pre-pubertal boar testes and in primary spermatocytes in post-pubertal boar testes. Therefore, SALL4 can be used as a stage-specific marker of developing germ cells in boar testes.

Knockdown of Sall4 inhibits intrahepatic cholangiocarcinoma cell migration and invasion in ICC-9810 cells.

In spite of improvements in surgical technology, the resectability and curability of intrahepatic cholangiocarcinoma (ICC) are still low. Our previous study showed that the strong Sal-like protein 4 (Sall4)-positive cases had shorter overall survival compared to Sall4-negative cases, indicating an oncogenic role of Sall4 in ICC. In this study, we aimed to explore the precise mechanism of Sall4 on ICC cell invasion and metastasis. We evaluated the expression of Sall4, PTEN, and Bmi-1 in 28 cases of adjacent tissues and 175 cases of ICC tissues by using immunohistochemical staining. We found that the expression of Sall4 and Bmi-1 was significantly increased in ICC tissues compared with the adjacent tissues, while PTEN expression was reduced in ICC tissues compared with the adjacent tissues, and there was a reverse relationship between Sall4 and PTEN in ICC, whereas there was a positive correlation in Sall4 and Bmi-1 expression in ICC. In addition, overall survival analysis showed that ICC patients with low PTEN exhibited a worse prognosis than ICC patients with high PTEN, and lower Bmi-1 expression showed a better prognosis than ICC patients with high Bmi-1. By a battery of experiments in vitro, we demonstrated that Sall4 promotes ICC cell proliferation, and progression of ICC might be through PTEN/PI3K/Akt and Bmi-1/Wnt/ß-catenin signaling and enhancing epithelial-mesenchymal transition process. Thus, Sall4 may be a potential target for the treatment of ICC metastasis.

Spermatogonial Nature of the Germ Cell Component of Canine Testicular Mixed Germ Cell-Sex Cord Stromal Tumours.

The present study has characterized the germ cell component of canine testicular mixed germ cell-sex cord stromal tumours (MGSCTs) by examining the histological nature and histochemical and immunohistochemical features using gonocytic and spermatogonial cellular markers, c-Kit, placental alkaline phosphatase (PLAP), protein gene product 9.5 (PGP9.5), Sal-like protein 4 (SALL4), and the periodic acid-Schiff (PAS) reaction. Histologically, all 45 examples of MGSCTs were classified as spermatocytic seminomas (SSs) and Sertoli cell tumours in combination. The germ cell component of all MGSCTs was negative by PAS staining. Immunohistochemically, PLAP immunoreactivity was lacking in the germ cell component of all MGSCTs, which is not consistent with a gonocytic origin. The germ cell component was positive for PGP9.5 and SALL4 in all MGSCTs and positive for c-Kit in 53% of MGSCTs, which is consistent with the phenotype of spermatogonia. Furthermore, the germ cell component in 71% of MGSCTs had moderate immunoreactivity for SALL4, which is suggestive of a spermatogonial phenotype. Conversely, 29% of cases had a minor population of germ cells showing strong SALL4 immunoreactivity, suggesting a phenotype similar to prespermatogonia. The results suggest that the germ cell component of canine MGSCTs is morphologically classified as SS, with the majority of cases showing the spermatogonial phenotype and some cases containing a small population of prespermatogonia.

Importance of SALL4 in the development and prognosis of hepatocellular carcinoma.

AIM: To detect the expression of sal-like protein 4 (SALL4) and to explore its relationship with clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC). METHODS: One hundred and twenty-six samples of HCC tissue, 44 of adjacent noncancerous cirrhotic tissue and 10 of liver hemangioma tissue, were obtained from patients who underwent hepatectomy for HCC at the Fourth Hospital of Hebei Medical University. None of the patients had received any form of treatment before the operation. After resection, all the tissues were fixed in 10% neutral formaldehyde and embedded in paraffin. Expression of SALL4 was detected by immunohistochemistry. Patients were followed up for postoperative survival until February 2014. The relationships between SALL4 expression level and clinicopathological data and prognosis of HCC were analyzed. RESULTS: SALL4 expression was negative in the 10 samples of tissue from liver hemangioma, was weakly positive in the two samples from adjacent noncancerous cirrhotic tissue, and positive in 58 samples of HCC tissues. The differences were statistically significant (P < 0.05). Expression of SALL4 was higher in patients with higher α-fetoprotein (AFP) levels, portal vein tumor thrombus, and later clinical stage based on the Barcelona Clinic Liver Cancer classification (P < 0.05). Among patients with negative expression, weakly positive expression, positive expression, and strongly positive expression of SALL4, the median survival time was 39, 25, 23, and 9 mo, respectively (P < 0.001). When both AFP and SALL4 were detected, patients who were negative for both AFP and SALL4, SALL4-positive only, AFP-positive only, and positive for both AFP and SALL4, had a median survival time of 41, 38, 31, and 12 mo, respectively (P < 0.001). CONCLUSION: Expression of SALL4 is relevant to the prognosis of HCC patients. Patients with higher expression levels of SALL4 and AFP have worse prognosis.

Expression of protein gene product 9.5 and Sal-like protein 4 in canine seminomas.

The aim of the present study was to characterize canine classical seminoma (SE) and spermatocytic seminoma (SS) by immunohistochemical expression of gonocytic and spermatogonial cellular markers (c-Kit, placental alkaline phosphatase [PLAP], protein gene product 9.5 [PGP9.5] and Sal-like protein 4 [Sall4]) and histochemically by the periodic acid-Schiff (PAS) reaction. Twenty-five cases of SE and 23 cases of SS were investigated. Two cases of dysgerminoma were also examined. c-Kit was expressed on the cell membrane of 13 of 25 cases of SE (52%) and four of 23 cases of SS (16%). This marker was not expressed in dysgerminoma. PLAP immunoreactivity was observed in the cytoplasm of neoplastic cells of six of 25 cases of SE (24%). PLAP was not expressed in cases of SS and dysgerminoma. All samples of SE, SS and dysgerminoma showed cytoplasmic expression of PGP9.5 and nuclear immunoreactivity for Sall4. There was fine granular cytoplasmic PAS staining in neoplastic cells in five of 25 cases of SE (20%), while all samples of SS and dysgerminoma cases were PAS negative. These findings suggest that it is not possible to differentiate canine SE and SS using these markers. This may be because canine SS may be derived from spermatogonia that can differentiate to spermatocytes and also because cases of canine SE might consist of neoplastic cells that have lost their gonocytic nature. This study was the first to show positive immunoreactivity for Sall4 in canine seminomas and dysgerminomas and expression of PGP9.5 in canine dysgerminomas.