Transcriptional Activity Of Sox9 Research Articles

SHP2 ablation mitigates osteoarthritic cartilage degeneration by promoting chondrocyte anabolism through SOX9.

Articular cartilage phenotypic homeostasis is crucial for life-long joint function, but the underlying cellular and molecular mechanisms governing chondrocyte stability remain poorly understood. Here, we show that the protein tyrosine phosphatase SHP2 is differentially expressed in articular cartilage (AC) and growth plate cartilage (GPC) and that it negatively regulates cell proliferation and cartilage phenotypic program. Postnatal SHP2 deletion in Prg4+ AC chondrocytes increased articular cellularity and thickness, whereas SHP2 deletion in Acan+ pan-chondrocytes caused excessive GPC chondrocyte proliferation and led to joint malformation post-puberty. These observations were verified in mice and in cultured chondrocytes following treatment with the SHP2 PROTAC inhibitor SHP2D26. Further mechanistic studies indicated that SHP2 negatively regulates SOX9 stability and transcriptional activity by influencing SOX9 phosphorylation and promoting its proteasome degradation. In contrast to published work, SHP2 ablation in chondrocytes did not impact IL-1-evoked inflammation responses, and SHP2's negative regulation of SOX9 could be curtailed by genetic or chemical SHP2 inhibition, suggesting that manipulating SHP2 signaling has translational potential for diseases of cartilage dyshomeostasis.

Abstract 2368: SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancers (TNBCs) are the most aggressive types of breast cancer, which lack the expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). TNBCs have a very poor prognosis and have few targeted therapy options. Therefore, the development of new TNBC treatment strategies is an urgent and unmet clinical need. Our previous studies have demonstrated that knockout of the SOX9 transcription factor decreased TNBC cell growth and tumor metastasis in vivo. Hypothesis: SOX9-binding proteins include upstream activators of this transcription factor which regulate SOX9 activity and control TNBC growth. Material and Methods: Immunoprecipitation (IP) in combination with mass spectrometry (IP-MS) analysis was used to identify SOX9 binding proteins, with support from MD Anderson’s proteomics core. The Mascot Score is a statistical score was used as the reference of targeted protein, and the data was summarized based on the Mascot Score. IP-Western Blotting analysis was used to confirm that the identified proteins bound SOX9 in TNBC cells. RNA-Seq analysis was used to identify SOX9 regulated genes in MDA MBA-231 and MDA MB-468 cells after SOX9 knockdown or in MCF-7 after SOX9 overexpression, with support from MD Anderson’s genomic biostatistics core. We then treated TNBC cells with or without siRNAs of these identified proteins to evaluate their effect on SOX9-regulated gene expression and TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. SOX9 transcriptional activity was measured using a reporter gene activity assay, and qRT-PCR assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated. Results: Using immunoprecipitation in combination with mass spectrometry (IP-MS) analysis, we identified multiple SOX9 binding proteins. Among them, proteins that demonstrated high Mascot Scores include Valosin-Containing Protein (VCP), protein S100A10 (S100A10); Caveolin 1 (CAV1). Knockdown of each of these SOX9-binding proteins (VCP, S100A10, or CAV1) decreased TNBC cell growth in vitro. Using mRNA-Seq analysis, we next identified 38 genes down-regulated by SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Using these genes as markers of SOX9 transcriptional activity, all three of these SOX9-binding proteins regulate SOX9 activity. Thus, VCP, CAV1, and S100A10 all are potential upstream proteins of SOX9 that can be targeted to inhibit TNBC growth. Conclusion: Our results demonstrate that VCP, S100A10, and CAV1, are SOX9-binding proteins that regulate SOX9 activity and control TNBC cell growth. Implications: These proteins represent potential targets for the treatment of TNBCs. Citation Format: Yanxia Ma, David H. Hawke, Ganiraju Manyam, Wenyi Wang, Abhijit Mazumdar, Powel Brown. SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2368.

RCC2 Promotes Esophageal Cancer Growth by Regulating Activity and Expression of the Sox2 Transcription Factor

Regulator of chromosome condensation 2 (RCC2) is a protein located in the centrosome, which ensures that cell division proceeds properly. Previous reports show that RCC2 is overexpressed in some cancers and could play a key role in tumor development, but the mechanisms concerning how this occurs are not understood. Furthermore, no evidence exists regarding its role in esophageal cancer. We studied the relevance of RCC2 in esophageal cancer growth and its regulation on Sox2, an important transcription factor promoting esophageal cancer. RCC2 was overexpressed in esophageal tumors compared with normal tissue, and this overexpression was associated with tumorigenicity by increasing cell proliferation, anchorage-independent growth, and migration. These oncogenic effects were accompanied by overexpression of Sox2. RCC2 upregulated and stabilized Sox2 expression and its target genes by inhibiting ubiquitination-mediated proteasome degradation. Likewise, RCC2 increased the transcriptional activity and promoter binding of Sox2. In vivo studies indicated that RCC2 and Sox2 were overexpressed in esophageal tumors compared with normal tissue, and this upregulation occurs in the esophageal basal cell layer for both proteins. In conditional knockout mice, RCC2 deletion decreased the tumor nodule formation and progression in the esophagus compared with wild-type mice. Proliferating cell nuclear antigen expression, a cell proliferation marker, was also downregulated in RCC2 knockout mice. Overall, our data show for the first time that RCC2 is an important protein for the stabilization and transcriptional activation of Sox2 and further promotion of malignancy in esophageal cancer. IMPLICATIONS: This study shows that RCC2 controls Sox2 expression and transcriptional activity to mediate esophageal cancer formation.

NF‐YA promotes the cell proliferation and tumorigenic properties by transcriptional activation of SOX2 in cervical cancer

NF‐YA is considered as a crucial regulator for the maintenance of cancer stem cell (CSC) and involved in various types of malignant tumours. However, the exact function and molecular mechanisms of NF‐YA in the progression of cervical cancer remains poorly understood. Here, the expression of NF‐YA detected by immunohistochemistry was gradually increased from normal cervical tissues, to the high‐grade squamous intraepithelial lesions, and then to cervical cancer tissues. NF‐YA promoted the cell proliferation and tumorigenic properties of cervical cancer cells as well as tumorsphere formation and chemoresistance in vitro. The luciferase reporter assay combined with mutagenesis analyses and Western blotting showed that NF‐YA trans‐activated the expression of SOX2 in cervical cancer. Furthermore, quantitative chromatin immunoprecipitation (qChIP) and electrophoretic mobility shift assay (EMSA) confirmed that NF‐YA protein directly bound to the CCAAT box region located upstream of the SOX2 promoter. Together, our data demonstrated that NF‐YA was highly expressed in cervical cancer and promoted the cell proliferation, tumorigenicity and CSC characteristic by trans‐activating the expression of SOX2.

Abstract 2469: RCC2 promotes esophageal cancer growth by regulating activity and expression of the Sox2 transcription factor

Abstract RCC2 is a protein located in the centrosome, which ensures that cell division proceeds properly. Previous reports show that RCC2 is overexpressed in some cancers and could play a key role in tumor development, but the mechanisms concerning how this occurs are not understood. Further, no evidence exists regarding its role in esophageal cancer. We studied the relevance of RCC2 in esophageal cancer growth and the mechanisms explaining how this process occurs. RCC2 was highly expressed in mouse and human tissues as well as esophageal cancer cell lines, and this overexpression was associated with tumorigenicity. RCC2 promoted cell proliferation, anchorage-independent growth, and migration. These oncogenic effects were accompanied by overexpression of Sox2, an important transcription factor promoting esophageal cancer. RCC2 upregulated and stabilized Sox2 expression by inhibiting proteasome degradation. Likewise, RCC2 increased the transcriptional activity and promoter binding of Sox2. In vivo studies indicated that RCC2 and Sox2 were overexpressed in esophageal tumors compared to normal controls, and this upregulation occurs in the basal cell layer for both proteins. In conditional knockout mice, RCC2 deletion decreased the tumor nodule formation and progression in the esophagus compared with “wild type” mice. PCNA expression, a cell proliferation marker, was also downregulated in RCC2 knockout mice. Overall, our data show for the first time that RCC2 is an important protein for the stabilization and transcriptional activation of Sox2 and further promotion of malignancy in cancer. Citation Format: Ali J. Calderon-Aparicio, Hiroyuki Yamamoto, Humberto de vitto, Tianshun Zhang, Qiushi Wang, Ann M. Bode, Zigang Dong. RCC2 promotes esophageal cancer growth by regulating activity and expression of the Sox2 transcription factor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2469.

PPARδ Interacts with the Hippo Coactivator YAP1 to Promote SOX9 Expression and Gastric Cancer Progression.

Despite established functions of PPARδ in lipid metabolism and tumorigenesis, the mechanisms underlying its role in gastric cancer are undefined. Here, we demonstrate that SOX9 was dramatically induced by stably expressing PPARδ and by its agonist GW501516 in human gastric cancer cell lines. PPARδ knockdown in patient-derived gastric cancer cells dramatically reduced SOX9 expression and transcriptional activity, with corresponding decreases in invasion and tumor sphere formation. Mechanistically, PPARδ induced SOX9 transcription through direct interaction with and activation of the Hippo coactivator YAP1. PPARδ-YAP1 interaction occurred via the C-terminal domain of YAP1, and both TEAD- and PPARE-binding sites were required for SOX9 induction. Notably, CRISPR/Cas9-mediated genetic ablation of YAP1 or SOX9 abolished PPARδ-mediated oncogenic functions. Finally, expression of PPARδ, YAP1, and SOX9 were significantly correlated with each other and with poor survival in a large cohort of human gastric cancer tissues. Thus, these findings elucidate a novel mechanism by which PPARδ promotes gastric tumorigenesis through interaction with YAP1 and highlights the PPARδ/YAP1/SOX9 axis as a novel therapeutic target in human gastric cancer. IMPLICATIONS: Our discovery of a new model supports a distinct paradigm for PPARδ and a crucial oncogenic function of PPARδ in gastric cancer through convergence on YAP1/TEAD signaling. Therefore, PPARδ/YAP1/SOX9 axis could be a novel therapeutic target that can be translated into clinics.

The Hippo effector TAZ promotes cancer stemness by transcriptional activation of SOX2 in head neck squamous cell carcinoma

The Hippo-TAZ signaling has emerged as a fundamental regulator underlying cancer stem cells (CSCs) stemness which intricately associates with local recurrence and metastatic spreading in head neck squamous cell carcinoma (HNSCC). However, the precise downstream targets of TAZ responsible for HNSCC CSCs maintenance remain largely underexplored. Here, we identified Sex determining region Y box 2 (SOX2) as a putative downstream target of TAZ to promote CSCs maintenance and tumorigenicity in HNSCC. Both TAZ and SOX2 were significantly enriched in CSCs subpopulation (CD44+CD133+) isolated from Cal27 and Fadu cells via fluorescence-activated cell sorting. TAZ knockdown significantly reduced expression of SOX2 at both mRNA and protein levels, whereas its ectopic overexpression markedly increased its abundance in HNSCC cells. Moreover, reintroduction of ectopic SOX2 abolished, at least in part, the reduced tumorsphere formation and tumorigenicity in vivo induced by TAZ knockdown. Mechanistically, transcriptional complex formed by TAZ and TEAD4 was recruited to two binding sites in SOX2 promoter, which in turn facilitated transcription of SOX2 in HNSCC cells. In addition, the abundance of TAZ and SOX2 was positively correlated in HNSCC clinical samples, and both upregulations of TAZ and SOX2 associated with the worst survival. Taken together, our data reveal a previously unknown mechanistic linkage between TAZ and SOX2 and identify SOX2 as a direct downstream target of TAZ in modulating CSCs self-renewal and maintenance in HNSCC. These findings suggest that targeting TAZ-SOX2 axis might be a promising therapeutic strategy for HNSCC.

Abstract 4498: O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer

Abstract Background: Pancreatic Cancer is an extremely aggressive disease with a bad outcome owing to its late detection, high rate of metastasis and self-renewal post-surgical and chemotherapeutic intervention. In cancer the balance between differentiation and self-renewal is tightly regulated by SOX2. Over expression of SOX2, as in pancreatic cancer, can tilt the balance towards self-renewal and thus increase the population of these undifferentiated “stem cells”. Methods: A CRISPR based OGT knockout kit was purchased from Origene and a knockout cell line was generated with S2VP10 cells. For the tumor initiation studies OGTi (OGT knockout cells) were implanted subcutaneously at a limiting dilution from 500,000 cells to 500 cells. Site directed mutagenesis was performed using theQuick-change Lightning from Agilent. Results: In the current study we show for the first time that SOX2 undergoes a type of post translational modification known as O GlcNAcylation. We also show for the first time that in humans this modification happens at Serine 246 and mutating this particular site via a side directed mutagenesis leads to a loss of O GlcNAcylation of SOX2.O GlcNAc modification of SOX2 further leads to its activation which then leads to its stabilization in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells. Conclusion: Our study highlights for the first time that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer which can be targeted therapeutically. Citation Format: Nikita S. Sharma, Vineet K. Gupta, Patricia Dauer, Roey Hadad, Kousik K. kesh, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4498.

Changes in SOX9 and RUNX2 protein activity correlate to the health state of human primary chondrocytes

Purpose: SOX9 and RUNX2 are the master transcription factors involved in cartilage and bone development respectively. Altered SOX9 and RUNX2 activity is implicated in osteoarthritis (OA) pathophysiology and hypertrophic differentiation of chondrocytes. We have previously shown that SOX9 and RUNX2 protein activity can be measured by Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP). To decipher the underlying signaling mechanism in OA pathophysiology, we aim to use TF-FRAP to correlate SOX9 and RUNX2 mobility to the health state of the chondrocytes. We hypothesize that the mobility of SOX9 increases with OA progression, while the mobility of RUNX2 decreases in chondrocyte hypertrophy. We measured the mobility and correlated protein activity of these transcription factors at the single cell level by TF-FRAP in healthy, preserved and OA chondrocytes. Methods: Healthy human primary chondrocytes (hPCs) were purchased from Articular Engineering, USA. Human preserved and OA hPCs were isolated from patients undergoing total knee replacement therapy. hPCs were seeded on glass coverslips and transfected with either SOX9-mGFP or eGFP-RUNX2. TF-FRAP was performed in at least 40 cells per condition in two healthy donors and three donors for each preserved and OA chondrocytes. TF-FRAP measurements were done using a Nikon A1 confocal microscope (Japan). In TF-FRAP, a small circular region (Ø 25 pixels) of the nucleus was bleached using a high intensity laser and the recovery of the fluorescence in the bleach spot is recorded. For every TF-FRAP measurement, 25 pre-bleach images and 260 post-bleach images were acquired at 4 frames per second for 60 seconds, with a frame size of 256x256 pixels. Higher immobile fraction (i.e., the fraction of transcription factors bound to DNA) and higher half-time recovery (longer DNA interaction times) were associated with a higher transcriptional activity. For all donors, early passage (≤3) cells were used for TF-FRAP and qPCR measurements. To correlate protein mobility and DNA binding with the protein activity, we quantified the expression levels of SOX9 target genes (ACAN and COL2A) and RUNX2 target genes (MMP13 and COL10) by qPCR. Results: In the healthy hPCs, the SOX9 immobile fraction and half-time recovery were 60% (SD ±6%) and 14.2 sec (SD ±3.2 sec) respectively. In preserved hPCs, the SOX9 immobile fraction and half-time to recover was significantly lower, ranging from 50% - 44% (SD ±7% - ±9.7%), and 14.2 - 13.3 sec (SD ±3.9 - ±4.5 sec) respectively. In OA hPCs, the SOX9 immobile fraction and half-time recovery was significantly lower than in the healthy and preserved hPCs, ranging from 48.6% - 42.8% (SD ±7.3% - ±8.4%), and half-time to recover ranged from 13.3 - 12.2 sec (SD ±3.9 - ±5.3 sec). RUNX2 immobile fraction and half-time to recover data spread was large in healthy, preserved and OA hPCs. In healthy hPCs, immobile fraction and half-time to recover were ranging from 33.6% - 47.9% (SD 19.4% - 20.7%) and 13.97 sec - 14.46 sec (SD 7.9 - 10.1 sec) respectively. In the preserved hPCs, the immobile fraction and half-time to recover were ranging from 38.8% - 47.0 % (SD 19.7% - 22.2%) and 11.7 sec - 14.8 sec (SD 6.8 - 8.2 sec) respectively. In OA hPCs, the immobile fraction and half-time to recover ranged from 33.0% - 41.02% (SD 20.3% - 25.7%) and 11.2 sec - 12.9 sec (SD 7.4 - 9.1 sec) respectively. We have an initial non-quantitative indication for a distinct cell population that, irrespective of the health state, shows decreased RUNX2 mobility, which could be indicative of a hypertrophic cell state. We will show the quantification and segregation. RT-qPCR measurements show decreased SOX9 and increased RUNX2 target gene expression during OA progression. Conclusions: Our data show that the SOX9 and RUNX2 transcriptional activity is differentially regulated depending on the health state of hPCs. If we assume a correlation between mobility and transcription factor activity, then we can say that SOX9 protein activity decreases with increasing OA progression. RUNX2 mobility data showed large spread especially in the preserved and OA donors possibly due to cells were in the different health state in the OA joint. Our findings at the single cell level provide novel insights into SOX9 and RUNX2 protein activity with respect to OA progression.

CDK1 Interacts with Sox2 and Promotes Tumor Initiation in Human Melanoma.

: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.

Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity

Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.

SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using “Cre-loxP”-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

SOX9 transcriptional activity is differentially regulated in healthy and osteoarthritic chondrocytes

SOX9 transcriptional activity is differentially regulated in healthy and osteoarthritic chondrocytes

Generation of a biotinylatable Sox2 mouse model to identify Sox2 complexes in vivo

Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to ‘complex-specific’ DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues.

SOX9 activity is induced by oncogenic Kras to affect MDC1 and MCMs expression in pancreatic cancer.

SRY (sex determining region Y)-box 9 (SOX9) is required for oncogenic Kras-mediated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasias (PanINs) and ultimately pancreatic ductal adenocarcinoma (PDAC). However, how oncogenic Kras affects SOX9 activity is not yet understood, and SOX9-associated genes in PDAC are also unknown at all. Here, we investigated the mechanistic link between SOX9 and oncogenic Kras, studied biological function of SOX9, and identified SOX9-related genes and their clinical significance in patients with PDAC. Our studies reveal that oncogenic Kras induces SOX9 mRNA and protein expression as well as phosphorylated SOX9 expression in human pancreatic ductal progenitor cells (HPNE) and pancreatic ductal cells (HPDE). Moreover, oncogenic Kras promoted nuclear translocation and transcriptional activity of SOX9 in these cells. TAK1/IκBα/NF-κB pathway contributed to induction of SOX9 by oncogenic Kras, and SOX9 in turn enhanced NF-κB activation. SOX9 promoted the proliferation of HPNE and PDAC cells, and correlated with minichromosome maintenance complex components (MCMs) and mediator of DNA damage checkpoint 1 (MDC1) expression. The overexpressive MDC1 was associated with less perineural and lymph node invasion of tumors and early TNM-stage of patients. Our results indicate that oncogenic Kras induces constitutive activation of SOX9 in HPNE and HPDE cells, and Kras/TAK1/IκBα/NF-κB pathway and a positive feedback between SOX9 and NF-κB are involved in this inducing process. SOX9 accelerates proliferation of cells and affects MCMs and MDC1 expression. MDC1 is associated negatively with invasion and metastasis of PDAC.

THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

SOX9 transcriptional activity is dependent on the chondrocyte health state and is directly regulated by WNT3A, IL1β and BMP7

SOX9 transcriptional activity is dependent on the chondrocyte health state and is directly regulated by WNT3A, IL1β and BMP7

The dark side of SOX2: cancer - a comprehensive overview.

The pluripotency-associated transcription factor SOX2 is essential during mammalian embryogenesis and later in life, but SOX2 expression can also be highly detrimental. Over the past 10 years, SOX2 has been shown to be expressed in at least 25 different cancers. This review provides a comprehensive overview of the roles of SOX2 in cancer and focuses on two broad topics. The first delves into the expression and function of SOX2 in cancer focusing on the connection between SOX2 levels and tumor grade as well as patient survival. As part of this discussion, we address the developing connection between SOX2 expression and tumor drug resistance. We also call attention to an under-appreciated property of SOX2, its levels in actively proliferating tumor cells appear to be optimized to maximize tumor growth - too little or too much SOX2 dramatically alters tumor growth. The second topic of this review focuses on the exquisite array of molecular mechanisms that control the expression and transcriptional activity of SOX2. In addition to its complex regulation at the transcriptional level, SOX2 expression and activity are controlled carefully by microRNAs, long non-coding RNAs, and post-translational modifications. In the Conclusion and Future Perspectives section, we point out that there are still important unanswered questions. Addressing these questions is expected to lead to new insights into the functions of SOX2 in cancer, which will help design novels strategies for more effectively treating some of the most deadly cancers.

Continuous hydrostatic pressure induces differentiation phenomena in chondrocytes mediated by changes in polycystins, SOX9, and RUNX2.

The present study aimed to investigate the long-term effects of hydrostatic pressure on chondrocyte differentiation, as indicated by protein levels of transcription factors SOX9 and RUNX2, on transcriptional activity of SOX9, as determined by pSOX9 levels, and on the expression of polycystin-encoding genes Pkd1 and Pkd2. ATDC5 cells were cultured in insulin-supplemented differentiation medium (ITS) and/or exposed to 14.7kPa of hydrostatic pressure for 12, 24, 48, and 96h. Cell extracts were assessed for SOX9, pSOX9, and RUNX2 using western immunoblotting. The Pkd1 and Pkd2 mRNA levels were detected by real-time PCR. Hydrostatic pressure resulted in an early drop in SOX9 and pSOX9 protein levels at 12h followed by an increase from 24h onwards. A reverse pattern was followed by RUNX2, which reached peak levels at 24h of hydrostatic pressure-treated chondrocytes in ITS culture. Pkd1 and Pkd2 mRNA levels increased at 24h of combined hydrostatic pressure and ITS treatment, with the latter remaining elevated up to 96h. Our data indicate that long periods of continuous hydrostatic pressure stimulate chondrocyte differentiation through a series of molecular events involving SOX9, RUNX2, and polycystins-1, 2, providing a theoretical background for functional orthopedic mechanotherapies.

Silibinin suppresses NPM-ALK, potently induces apoptosis and enhances chemosensitivity in ALK-positive anaplastic large cell lymphoma

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion protein carrying constitutively active tyrosine kinase, is known to be central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK+ALCL). Here, it is reported that silibinin, a non-toxic naturally-occurring compound, potently suppressed NPM-ALK and effectively inhibited the growth and soft agar colony formation of ALK+ALCL cells. By western blots, it was found that silibinin efficiently suppressed the phosphorylation/activation of NPM-ALK and its key substrates/downstream mediators (including STAT3, MEK/ERK and Akt) in a time- and dose-dependent manner. Correlating with these observations, silibinin suppressed the expression of Bcl-2, survivin and JunB, all of which are found to be upregulated by NPM-ALK and pathogenetically important in ALK+ALCL. Lastly, silibinin augmented the chemosensitivity of ALK+ALCL cells to doxorubicin, particularly the small cell sub-set expressing the transcriptional activity of Sox2, an embryonic stem cell marker. To conclude, the findings suggest that silibinin might be useful in treating ALK+ALCL.