AKT-mediated phosphorylation of Sox9 induces Sox10 transcription in a murine model of HER2-positive breast cancer

Background Approximately 5–10% of HER2-positive breast cancers can be defined by low expression of the Ste20-like kinase, SLK, and high expression of SOX10. Our lab has observed that genetic deletion of SLK results in the induction of Sox10 and significantly accelerates tumor initiation in a HER2-induced mammary tumor model. However, the mechanism responsible for the induction of SOX10 gene expression in this context remains unknown. Methods Using tumor-derived cell lines from MMTV-Neu mice lacking SLK and biochemical approaches, we have characterized the signaling mechanisms and relevant DNA elements driving Sox10 expression. Results Biochemical and genetic analyses of the SOX10 regulatory region in SLK-deficient mammary tumor cells show that Sox10 expression is dependent on a novel −7kb enhancer that harbors three SoxE binding sites. ChIP analyses demonstrate that Sox9 is bound to those elements in vivo. Our data show that AKT can directly phosphorylate Sox9 in vitro at serine 181 and that AKT inhibition blocks Sox9 phosphorylation and Sox10 expression in SLK(-/-) tumor cells. AKT-mediated Sox9 phosphorylation increases its transcriptional activity on the Sox10 −7kb enhancer without altering its DNA-binding activity. Interestingly, analysis of murine and human mammary tumors reveals a direct correlation between the levels of active phospho-Sox9 S181 and Sox10 expression. Conclusions Our results have identified a novel Sox10 enhancer and validated Sox9 as a direct target for AKT. As Sox10 is a biomarker for triple-negative breast cancers (TNBC), these findings might have major implications in the targeting and treatment of those cancers.

SOX11, SOX10 and MITF Gene Interaction: A Possible Diagnostic Tool in Malignant Melanoma

Malignant melanoma (MM) is a highly heterogenic tumor whose histological diagnosis might be difficult. This study aimed to investigate the diagnostic and prognostic utility of the conventional pan-melanoma cocktail members (HMB-45, melan-A and tyrosinase), in conjunction with SOX10 and SOX11 immunohistochemical (IHC) expression. In 105 consecutive cases of MMs and 44 of naevi, the IHC examination was performed using the five-abovementioned markers, along with microphthalmia transcription factor (MITF), S100, and Ki67. Correlation with the clinicopathological factors and a long-term follow-up was also done. Survival analysis was performed with Kaplan–Meier curves and compared with TCGA public datasets. None of the 44 naevi expressed SOX11, but its positivity was seen in 52 MMs (49.52%), being directly correlated with lymphovascular invasion, the Ki67 index, and SOX10 expression. HMB-45, SOX10, and tyrosinase, but not melan-A, proved to differentiate the naevi from MMs successfully, with high specificity. Triple MITF/SOX10/SOX11 co-expression was seen in 9 out of 15 negative conventional pan-melanoma-cocktail cases. The independent prognostic value was proved for the conventional pan-melanoma cocktail (triple positivity for HMB-45, melan-A, and tyrosinase) and, independently for HMB-45 and tyrosinase, but not for melan-A, SOX10, or SOX11. As consequence, to differentiate MMs from benign naevi, melan-A should be substituted by SOX10 in the conventional cocktail. Although the conventional pan-melanoma cocktail, along with S100 can be used for the identification of melanocytic origin of tumor cells and predicting prognosis of MMs, the conventional-adapted cocktail (triple positivity for HMB-45, SOX10, and tyrosinase) has a slightly higher diagnostic specificity. SOX11 can be added to identify the aggressive MMs with risk for lymphatic dissemination and the presence of circulating tumor cells.

Rapid and Efficient Generation of Myelinating Human Oligodendrocytes in Organoids

Human stem cell derived brain organoids are increasingly gaining attention as an ideal model system for investigating neurological diseases, particularly those that involve myelination defects. However, current protocols for generating brain organoids with sufficiently mature oligodendrocytes that deposit myelin on endogenously produced neurons are lengthy and complicated. Taking advantage of a human pluripotent stem cell line that reports on SOX10 expression, we developed a protocol that involves a 42 day exposure of neuroectoderm-derived organoids to a cocktail of growth factors and small molecules that collectively foster oligodendrocyte specification and survival. Importantly, the resulting day 42 brain organoids contain both myelinating oligodendrocytes, cortical neuronal cells and astrocytes. These oligodendrocyte brain organoids therefore constitute a valuable and tractable platform for functional neurogenomics and drug screening for white matter diseases.

Glomus coccygeum in pilonidal sinus surgical specimens: report of two rare cases with special reference to SOX10 expression

We report two new cases of glomus coccygeum in pilonidal sinus excision specimens. The positive expression of glomus coccygeum cells for SOX10 is used for the first time. SOX10 is a useful immunohistochemical marker for identifying this microanatomical structure, confirming the diagnosis and may help the differential diagnosis. The glomus coccygeum cells are probably neural crest-derived from multipotent Schwann cell precursors.

AKT-mediated phosphorylation of Sox9 induces Sox10 transcription in a murine model of HER2-positive breast cancer.

Background: Approximately 5-10% of HER2-positive breast cancers can be defined by low expression of the Ste20-like kinase, SLK, and high expression of SOX10. Our lab has observed that genetic deletion of SLK results in the induction of Sox10 and significantly accelerates tumor initiation in a HER2-induced mammary tumor model. However, the mechanism responsible for the induction of SOX10 gene expression in this context remains unknown.Methods: Using tumor derived cell lines from MMTV-Neu mice lacking SLK and biochemical approaches, we have characterized the signaling mechanisms and relevant DNA elements driving Sox10 expression. Results: Biochemical and genetic analyses of the SOX10 regulatory region in SLK-deficient mammary tumor cells show that Sox10 expression is dependent on a novel -7kb enhancer that harbors three SoxE binding sites. ChIP analyses demonstrate that Sox9 is bound to those elements in vivo. Our data show that AKT can directly phosphorylate Sox9 in vitro at serine 181 and that AKT inhibition blocks Sox9 phosphorylation and Sox10 expression in SLK(-/-) tumor cells. AKT-mediated Sox9 phosphorylation increases its transcriptional activity on the Sox10 -7kb enhancer without altering its DNA binding activity. Interestingly, analysis of murine and human mammary tumors reveals a direct correlation between the levels of active phospho-Sox9 S181 and Sox10 expression.Conclusions: Our results have identified a novel Sox10 enhancer and validated Sox9 as direct target for AKT. As Sox10 is a biomarker for triple negative breast cancers (TNBC), these findings might have major implications in the targeting and treatment of those cancers.

Immunohistochemical determination of PD-L1 expression with three different antibody clones and determination of SOX10 expression in triple negative breast cancer.

e15237 Background: In triple negative breast cancer (TBNC), checkpoint inhibitors directed against PD-L1/PD-1 show an improvement of therapy. For the immunohistochemical diagnosis of the predictive biomarker PD-L1, there are various antibodies and kits available. We examined the expression of PD-L1 in TNBC with different antibody clones to compare these results with regard to the staining of tumor cell membranes, staining of immune cells and cytoplasmic staining in order to find out whether the different clones or methods can be exchanged. It was also checked whether PD-L1 or SOX10 expression correlates with the existing clinical parameters. Methods: Breast cancer tissue of 60 patients with TNBC were examined for the expression of PD-L1 and SOX10 by immunohistochemistry. The detection kit used, was the ultraView universal alkaline phosphatase red detection kit for the antibodies anti-human PD-L1 clone 22C3 from Dako, the clone 28-8 from abcam and the SOX10 antibody clone SP267 from Cell Marque. The anti-PD-L1 antibody clone SP142 was detected with OptiView DAB. The cut-offs for the expression of PD-L1 at the tumor cell membrane were < 1%, > 1 to < 50% and > 50%. In the evaluation of SP142 the staining of the immune cells was evaluated with the score: percentage of PD-L1 positive immune cells to the tumor cells that were present. For SOX10, the nuclear staining was evaluated with the score: < 1, > 1 to 50, > 50 to > 100 and 100. The relationship between PD-L1 expression (TC and IC) and SOX10 expression was evaluated with the clinical parameters of the patients from the time of diagnosis until the end of data collection. Results: The antibodies clone 22C3 and clone 28-8 lead to the same results (22,0 % PD-L1 (TC) positive). Clone SP142 showed significantly (p < 0,001) more PD-L1 positive cases(40,7%). For the additional evaluation of the cytoplasmic staining with clone 22C3 and clone 28-8 it could be shown that the PD-L1 Expression is equivalent (40,7%) to the immune cell staining with clone SP142. With exception of Ki67 we were unable to demonstrate any correlation between PD-L1 (membrane and cytoplasmic), SOX10 and other clinical parameters in TBNC. Conclusions: The antibodies clone 22C3 and 28-8 can be used equivalently for PD-L1 determination in TBNC. Clone SP142 showed different results. The cytoplasmatic staining with 22C3 and 28-8 could gain importance in the future because the results were equivalent to the immune cell staining with clone SP142. There is no correlation between PD-L1 and SOX10 in TNBC.