SOX9 Protein Levels Research Articles

Abstract PR06: Molecular and functional interactions between AKT and SOX2 in breast carcinoma

Abstract The pluripotency-associated transcription factor SOX2 (Sex determining region Y - Box 2) is a key regulator of early organogenesis and embryonic stem cell fate. Supporting the notion that embryonic pathways may reactivate during oncogenesis, SOX2 expression was more recently documented also in several cancers. In breast carcinoma (BC), SOX2 expression is linked to the disease-initiating clonogenic cancer stem cell (CSC) compartment and indicative of disease aggressiveness. However, the molecular regulation of SOX2 in BC remains largely unknown. Here, we describe a functional dependence of SOX2 protein expression on the Ser/Thr-kinase AKT, a central mediator of cell growth, proliferation and metabolism, whose deregulation is a common determinant in BC. Using qRT-PCR and immunoblot analyses, we confirm SOX2 expression in a series of human BC cell lines and primary patient-derived samples, and more importantly, observe co-induction of SOX2 protein expression and AKT kinase activity in cultivation conditions favoring the stem cell compartment. Suggesting functional coupling, lentiviral-induced knockdown of SOX2 and specific AKT kinase inhibition jointly impair BC cell clonogenicity, as deduced from in vitro tumor spheres assays. Verifying SOX2 as a functional downstream target of AKT, ectopic expression of a mCherry-SOX2 fusion protein rescues spheres formation from AKT-inhibited BC cells in vitro and restores tumorigenicity in xenotransplantation assays in vivo. Moreover, AKT inhibitors effectively impair the growth of putative CSC, as identified by an active SOX2 regulatory region (SRR) reporter construct, while conventional chemotherapeutic drugs (e.g. cisplatin or paclitaxel) suppress growth but allow selective survival and thereby enrichment of this cell population. On the molecular level, Western analyses indicate a stringent correlation between SOX2 protein expression and AKT kinase activity in all BC cell lines and patient-derived samples tested. Importantly, the inhibition of either AKT itself (MK-2206, Akti1/2) or of its up-stream regulator PI3K (Wortmannin, GDC-0941) impair SOX2 expression, whereas a modulation of de no protein synthesis by either rapamycin or cycloheximide has only minor impact on SOX2 protein levels on the same time-span. Cell fractionation studies further illustrate a preferential cytoplasmic clearance of SOX2 protein in AKT-inhibited cells, which is largely restored by co-treatment with the proteasomal inhibitor bortezomib. Taken together, these data indicate that AKT regulates SOX2 protein expression via post-translational mechanisms in BC. Supporting this notion, an AKT-phosphorylation motif was identified within the nuclear localization sequence of SOX2 and a direct physical interaction of both proteins verified by co-immunoprecipitation. Confirming a modulation of SOX2 nuclear entry, we observe a temporal cytoplasmic retention of SOX2 in BC cells of stalled AKT-kinase activity. This signal retention is fully restored by co-treatment with leptomycin B, indicating that the observed cytoplasmic accumulation results from nuclear exit of pre-formed SOX2 protein under compromised entry conditions (i.e. persisting AKT kinase inhibition). At extended incubation times, the SOX2 signal gradually disappears in AKT-inhibited cells but can be rescued by concomitant treatment with bortezomib. Importantly, the restored SOX2 protein mislocalizes to the cytosol in anti-AKT and bortezomib co-treated cells, demonstrating that in the absence of AKT activity the rescued SOX2 protein cannot perform nuclear entry. Taken together, our data indicate an unrecognized coupling of AKT and SOX2 proteins that regulates clonogenicity and in vivo tumorigenicity in human BC. Our investigations reveal important mechanistic details about the regulation of SOX2 subcellular distribution and may provide valuable insights on the potential and limitations of PI3K/AKT/mTOR inhibitors currently used in clinical trials of BC. Citation Format: Thorsten Schaefer, Hui Wang, Perihan Mir, Martina Konantz, Tamara C. Pereboom, Britta Merz, Tanja Fehm, Sven Perner, Oliver K. Rothfuss, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. Molecular and functional interactions between AKT and SOX2 in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR06.

DDX17 (P72), a Sox2 binding partner, promotes stem-like features conferred by Sox2 in a small cell population in estrogen receptor-positive breast cancer

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy, we tested our hypothesis that Sox2, which is a key regulator of the RR phenotype, is under the control of its binding partners. In this study, we focused on DDX17, known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation, we confirmed the binding between DDX17 and Sox2, although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells, it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR, DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2, as well as 3 of its specific gene targets including MUC15, CCND1 and CD133. Correlating with these findings, siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude, DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells, DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes.

Elp3 drives Wnt-dependent tumor initiation and regeneration in the intestine.

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.

Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.

Expression of pluripotency markers in the bovine uterus with adenomyosis.

BackgroundAdenomyosis is a proliferative uterine dysfunction with unknown aetiology. One possible mechanism of its development involves disturbances in stem cell differentiation in uterine tissue. Previously, we identified pluripotent/multipotent cells in the bovine uterus, therefore our present study focused on determining expression of pluripotency markers, NANOG, OCT4 and SOX2, in bovine adenomyotic tissues and cells.FindingsImmunolocalisation revealed protein expression of NANOG, OCT4 and SOX2 in both normal and adenomyotic uteri. mRNA expression for NANOG and OCT4 was increased in tissues obtained from uteri with adenomyosis compared to controls, but at the protein level there were no significant differences. mRNA expression for all three pluripotency markers was higher in myometrial cells isolated from uteri with adenomyotic lesions than in those isolated from normal uteri. The protein level of NANOG and SOX2 was decreased in stromal cells from adenomyotic tissues, whereas the level of OCT4 and SOX2 was increased in myometrial cells obtained from dysfunctional uteri.ConclusionsThe results indicate significant changes in expression of pluripotency markers in adenomyotic compared to normal uteri, which suggest the involvement of uterine stem cells in adenomyosis.

Abstract 1957: Negative regulation of Sox9 by glycogen synthase kinase 3 beta phosphorylation and SCFFbw7-dependent ubiquitination in cancer

Abstract Sox9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of transcription factors, with known functions in proliferation, epithelial-mesenchymal transition (EMT), stem cell maintenance and senescence. Sox9 protein levels are elevated in several different cancer types, including lung cancer and osteosarcoma. Furthermore, Sox9 overexpression is associated with poor survival in both cancer types. We recently reported that Sox9 is a direct transcriptional target of the Notch signaling pathway and mediates Notch1-induced EMT in lung cancer (Capaccione et al., Oncotarget, 2014). Fbxw7 (F-box and WD repeat domain-containing 7) is a member of the SCFFbw7E3 ubiquitin ligase complexthat targets several oncogenic proteins for degradation, including Notch1. We hypothesized that Sox9 is targeted for degradation by SCFFbw7. Knocking down or overexpressingFbxw7 significantly increasedor decreased, respectively, endogenous Sox9 protein levels in lung cancer and osteosarcoma cell lines. In addition, overexpression of Fbxw7 resulted in active degradation of ectopic Sox9 protein levels, when assessed in combination with cycloheximide, and did so in a dose dependent-manner.Furthermore, both Sox9 and FBXW7 were detected in immunoblots after the reciprocal protein was immunoprecipitated. Because substrate degradation by SCFFbw7 is typically phosphorylation-dependent, frequently by glycogen synthase kinase (GSK)3-beta, we examined whether modulation of GSK3-beta could affect FBXW7-induced Sox9 degradation. Treatment of cells with the GSK3-beta inhibitor, CHIR99021, completely abolished Sox9 degradation by ectopically overexpressed FBXW7, in a dose-dependent manner. We next identified four putative phosphorylation sites within potential FBXW7 degron motifs in Sox9.Mass spectrophotometric analysis confirmed these sites within Sox9 are indeed phosphorylated.In summary, we found that the E3 ubiquitin ligase complex (SCFFbw7) targets Sox9 for ubiquitin-mediated degradation in a GSK3-beta-mediated Sox9 phosphorylation-dependent manner. As a result, this negative regulation of Sox9 by GSK3-beta/FBXW7 could lead to inhibition of Sox9-mediated proliferation, EMT and stemness. This new regulatory mechanism of Sox9 could have diagnostic and therapeutic implications for lung cancer, osteosarcoma, as well as other cancers. Citation Format: Xuehui Hong, Wenyu Liu, Hiroyuki Inuzuka, Lianxin Liu, Sharon R. Pine. Negative regulation of Sox9 by glycogen synthase kinase 3 beta phosphorylation and SCFFbw7-dependent ubiquitination in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1957. doi:10.1158/1538-7445.AM2015-1957

Loss of SOX2 expression induces cell motility via vimentin up-regulation and is an unfavorable risk factor for survival of head and neck squamous cell carcinoma

Recurrent gain on chromosome 3q26 encompassing the gene locus for the transcription factor SOX2 is a frequent event in human squamous cell carcinoma, including head and neck squamous cell carcinoma (HNSCC). Numerous studies demonstrated that SOX2 expression and function is related to distinct aspects of tumor cell pathophysiology. However, the underlying molecular mechanisms are not well understood, and the correlation between SOX2 expression and clinical outcome revealed conflicting data. Transcriptional profiling after silencing of SOX2 expression in a HNSCC cell line identified a set of up-regulated genes related to cell motility (e.g. VIM, FN1, CDH2). The inverse regulation of SOX2 and aforementioned genes was validated in 18 independent HNSCC cell lines from different anatomical sites. The inhibition of cell migration and invasion by SOX2 was confirmed by constant or conditional gene silencing and accelerated motility of HNSCC cells after SOX2 silencing was partially reverted by down-regulation of vimentin. In a retrospective study, SOX2 expression was determined by immunohistochemical staining on tissue microarrays containing primary tumor specimens of two independent HNSCC patient cohorts. Low SOX2 expression was found in 19.3% and 44.9% of primary tumor specimens, respectively. Univariate analysis demonstrated a statistically significant correlation between low SOX2 protein levels and reduced progression-free survival (Cohort I 51 vs. 16 months; Cohort II 33 vs. 12 months) and overall survival (Cohort I 150 vs. 37 months; Cohort II 33 vs. 16 months). Multivariate Cox proportional hazard model analysis confirmed that low SOX2 expression serves as an independent prognostic marker for HNSCC patients. We conclude that SOX2 inhibits tumor cell motility in HNSCC cells and that low SOX2 expression serves as a prognosticator to identify HNSCC patients at high risk for treatment failure.

Sex-determining region Y-related high mobility group box (SOX)-2 is overexpressed in cervical squamous cell carcinoma and contributes cervical cancer cell migration and invasion in vitro.

Sex-determining region Y-related high mobility group box 2 (SOX-2) is a key pluripotency-associated transcription factor and may be implicated in the pathogenesis of cervical squamous cell carcinoma (SCC). The aim of this study was to explore SOX-2 expression in cervical SCC tissues and to examine whether and how SOX-2 regulates the malignant behaviors of cervical SCC cells in vitro. We here found that SOX-2 expression in the examined cervical SCC tissues was higher than that in the normal cervical and cervical intraepithelial neoplasia (CIN) tissues. Higher protein level of SOX-2 (nuclear positive staining cells ≥50 %) was detected in 34.9 % (29 out of 83 cases) of cervical SCC patients. We also noted that 100 % of well-differentiated and 66.7 % of moderately differentiated cervical SCCs showed lower SOX-2 expression (nuclear positive staining cells <50 %), while 58.8 % of poorly differentiated tumors had higher SOX-2 expression (P < 0.05). Furthermore, the migratory and invasive capabilities of SiHa cervical cancer cells were enhanced when SOX-2 was upregulated whereas suppressed when SOX-2 was downregulated. Also, the phosphorylation levels of protein kinase B (Akt) and extracellular regulated protein kinases (ERK) 1/2 were increased in SOX-2-overexpressed cancer cells but decreased in SOX-2-depleted cells. Additionally, LY294002 (Akt pathway inhibitor) or U0126 (ERK pathway inhibitor) significantly suppressed SOX-2-overexpression-induced migration and invasion in SiHa cells. Our results indicate that differentially expressed SOX-2 is associated with tumor differentiation (P < 0.05) and that SOX-2 contributes to the migratory and invasive behaviors of cervical SCC in vitro.

Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.

The generation of differentiated and functional neurons is a complex process, which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200, miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells, induction of miR-200 family was observed in differentiating neural stem cells, demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However, inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells, an inverse relationship was observed between expression of reprogramming transcription factors (SOX2, KLF4, NANOG, OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover, we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion, our results indicate that increased expression of the miR-200 family promotes neuronal differentiation, while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4.

Extended Production of Cortical Interneurons into the Third Trimester of Human Gestation.

In humans, the developmental origins of interneurons in the third trimester of pregnancy and the timing of completion of interneuron neurogenesis have remained unknown. Here, we show that the total and cycling Nkx2.1(+)and Dlx2(+)interneuron progenitors as well as Sox2(+)precursor cells were higher in density in the medial ganglionic eminence (MGE) compared with the lateral ganglionic eminence and cortical ventricular/subventricular zone (VZ/SVZ) of 16-35 gw subjects. The proliferation of these progenitors reduced as a function of gestational age, almost terminating by 35 gw. Proliferating Dlx2(+)cells were higher in density in the caudal ganglionic eminence (CGE) compared with the MGE, and persisted beyond 35 gw. Consistent with these findings, Sox2, Nkx2.1, Dlx2, and Mash1 protein levels were higher in the ganglionic eminences relative to the cortical VZ/SVZ. The density of gamma-aminobutyric acid-positive (GABA(+)) interneurons was higher in the cortical VZ/SVZ relative to MGE, but Nkx2.1 or Dlx2-expressing GABA(+)cells were more dense in the MGE compared with the cortical VZ/SVZ. The data suggest that the MGE and CGE are the primary source of cortical interneurons. Moreover, their generation continues nearly to the end of pregnancy, which may predispose premature infants to neurobehavioral disorders.

Partial inhibition of differentiation associated with elevated protein levels of pluripotency factors in mouse embryonic stem cells expressing exogenous EGAM1N homeoprotein

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Effects of pulsed electromagnetic field on differentiation of HUES-17 human embryonic stem cell line.

Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

The effects of triclosan on pluripotency factors and development of mouse embryonic stem cells and zebrafish.

Triclosan (TCS) poses potential risks to reproduction and development due to its endocrine-disrupting properties. However, the mechanism of TCS's effects on early embryonic development is little known. Embryonic stem cells (ESC) and zebrafish embryos provide valuable models for testing the toxic effects of environmental chemicals on early embryogenesis. In this study, mouse embryonic stem cells (mESC) were acutely exposed to TCS for 24 h, and general cytotoxicity and the effect of TCS on pluripotency were then evaluated. In addition, zebrafish embryos were exposed to TCS from 2- to 24-h post-fertilization (hpf), and their morphology was evaluated. In mESC, alkaline phosphatase staining was significantly decreased after treatment with the highest concentration of TCS (50 μM). Although the expression levels of Sox2 mRNA were not changed, the mRNA levels of Oct4 and Nanog in TCS-treated groups were significantly decreased compared to controls. In addition, the protein levels of Oct4, Sox2 and Nanog were significantly reduced in response to TCS treatment. MicroRNA (miR)-134, an expression inhibitor of pluripotency markers, was significantly increased in TCS-treated mESC. In zebrafish experiments, after 24 hpf of treatment, the controls had developed to the late stage of somitogenesis, while embryos exposed to 300 μg/L of TCS were still at the early stage of somitogenesis, and three genes (Oct4, Sox2 and Nanog) were upregulated in treated groups when compared with the controls. The two models demonstrated that TCS may affect early embryonic development by disturbing the expression of the pluripotency markers (Oct4, Sox2 and Nanog).

288 HDAC-1 Inhibition Prevents Smoking Induced PanIN Lesions and EMT in Kras Mouse Model of Pancreatic Cancer

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

290 CUGBP1 and HuR Regulate E-Cadherin Translation by Altering Recruitment of E-Cadherin mRNA to Processing Bodies During Total Parenteral Nutrition

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

289 Tight Junction Protein Claudin-2 Is a Direct Target of Vitamin D Receptor Signaling in Intestinal Epithelium

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

Effect of Transforming Growth Factor‐β (TGF‐β) on Sox9 function and chondrocyte phenotype (LB40)

TGF‐β enhances Sox9 protein expression as well as downstream targets that regulate extracellular matrix production in bovine articular chondrocytes (BACs). The extracellular matrix protein 3‐Prime‐Phoshoadenosine 5‐Prime‐Phosphosulfate Synthase 2 (Papss2) has been associated with spondyloepimetaphyseal dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. Our work and that of others has shown that TGF‐β can regulate Papss2 expression in articular cartilage. We aim to identify whether Sox9 is involved downstream of TGF‐β in the regulation of Papss2 mRNA expression.Using primary BACs in micromass cultures, we analyzed the effect of TGF‐β (5ng/ml) on chondrocyte phenotype, Sox9 protein expression, stability, cellular localization and activity. We also assessed the effect of TGF‐β and/or Sox9 overexpression on the mRNA expression of Papss2, Prg4 and GAPDH via quantitative real‐time PCR.We found that treatment with TGF‐β enhanced proteoglycan content and delayed chondrocyte hypertrophic differentiation in BACs. We also observed an increase in Sox9 protein levels and stability, without changes in mRNA. The increase in Sox9 protein correlated with enhanced Col2a‐reporter activity. Importantly, we found Papss2 to be upregulated via Sox9 when wild‐type (WT)‐Sox9 was overexpressed via adenoviral transduction (50 MOI/cell). Interestingly, treatment +/‐ TGF‐β when Sox9 was overexpressed did not further increase Papss2 mRNA, supporting the hypothesis that Sox9 works downstream of TGF‐ β. However, TGF‐β, but not Sox9, increased Prg4 mRNA expression, suggesting specificity of signaling. A GAPDH control was examined and remained unchanged in response to either treatment. The regulation of Papss2 by Sox9 represents a novel paradigm involving TGF‐β‐dependent signaling in articular chondrocytes.Grant Funding Source: Supported by NIAMS‐ NIH

Abstract B17: Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma.

Abstract Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans. Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9. Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B17.

Inducible intestine-specific deletion of Krüppel-like factor 5 is characterized by a regenerative response in adult mouse colon

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.