SOX11 expression correlates to promoter methylation and regulates tumor growth in hematopoietic malignancies

<p>Abstract</p> <p>Background</p> <p>The transcription factor SOX11 plays an important role in embryonic development of the central nervous system (CNS) and is expressed in the adult immature neuron but is normally not expressed in any other adult tissue. It has recently been reported to be implicated in various malignant neoplasms, including several lymphoproliferative diseases, by its specific expression and in some cases correlation to prognosis. SOX11 has been shown to prevent gliomagenesis <it>in vivo </it>but the causes and consequences of aberrant expression of <it>SOX11 </it>outside the CNS remain unexplained.</p> <p>Results</p> <p>We now show the first function of <it>SOX11 </it>in lymphoproliferative diseases, by demonstrating <it>in vitro </it>its direct involvement in growth regulation, as assessed by siRNA-mediated silencing and ectopic overexpression in hematopoietic malignancies. Gene Chip analysis identified cell cycle regulatory pathways, including Rb-E2F, to be associated with SOX11-induced growth reduction. Furthermore, promoter analysis revealed that <it>SOX11 </it>is silenced through DNA methylation in B cell lymphomas, suggesting that its regulation is epigenetically controlled.</p> <p>Conclusions</p> <p>The data show that SOX11 is not a bystander but an active and central regulator of cellular growth, as both siRNA-mediated knock-down and ectopic overexpression of <it>SOX11 </it>resulted in altered proliferation. Thus, these data demonstrate a tumor suppressor function for <it>SOX11 </it>in hematopoietic malignancies and revealed a potential epigenetic regulation of this developmentally involved gene.</p

S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL

The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

<p>Abstract</p> <p>Background</p> <p>The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking.</p> <p>Methods</p> <p>SOX11 expression and clinicopathological data was compared using χ²test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation.</p> <p>Results</p> <p>SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5'-Aza-2'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11.</p> <p>Conclusions</p> <p>SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours.</p

Functional studies of SOX11 and T-STAR - Translation of tumor-associated genes into clinical and biological insight

Cancer is a disease that will affect most of us during a lifetime. Although the survival of cancer patients has incresed due to better treatment and earlier diagnosis, the prevalance of the disease is still increasing. This increase in the number of cancer cases diagnosed will impose a challenge to the cancer care and higher costs for socitey. Personalized medicine aims at reducing these costs while at the same time improving the care for the patient, mainly by using a more individualized therapy, but also a more preventive cancer care and even earlier diagnosis. For example, a more indivivualized treatment can target specific genetic aberrations found in a patient, and avoid treating those patients who lack the aberrations and therefore will not respond. To accomplish the goals of personalized medicine, biomarkers that can divide patients into different subgroups are needed. The aim of this thesis was to explore SOX11 and T-STAR as potential biomarkers in cancer. I was also interested in the function of these proteins in the tumor tissues where they are specifically expressed. Immunohistochemistry on tissue microarrays was used to evaluate the expression of SOX11 and T-STAR in primary tumor tissues, and the expression was then correlated to survival in the patients. The function of the proteins was explored using RNA interference and overexpression experiements in cancer cell lines. The results from my studies are presented in five papers in this thesis. First, we could confirm the diagnostic role of SOX11 in mantle cell lymphoma (MCL). Second, I show the prognostic potential of this protein, as patients with high expression of SOX11 are associated with a better survival, both in MCL and high-grade epithelial ovarian cancer. In MCL, SOX11 could possibly complement the MCL international prognostic index MIPI , a prognostic biomarker already in use in this disease. Third, in both tumor types SOX11 functions as a tumor-suppressor in vitro. Regarding T-STAR, its expression is correlated with better outcome for breast cancer patients, and therefore shows potential as a prognostic biomarker. Our studies also show that T-STAR has a growth-inhibitory function in breast cancer cell lines. In conclusion, both SOX11 and T-STAR are biomarkers that could be used in personalized medicine to subgroup patients and potentially guide treatment decisions. Both proteins function as growth-inhibitors in vitro, which correlates to the better prognosis for the patients with tumors expressing the proteins in vivo

Nuclear T-STAR Protein Expression Correlates with HER2 Status, Hormone Receptor Negativity and Prolonged Recurrence Free Survival in Primary Breast Cancer and Decreased Cancer Cell Growth In Vitro.

T-STAR (testis-signal transduction and activation of RNA) is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i) survival analysis of patients with primary invasive breast cancer and (ii) experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC) in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS) and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification

T-STAR nuclear and cytoplasmic expression in relation to patient- and tumor characteristics in the total cohort (χ2 test for linear trend).

<p>T-STAR nuclear and cytoplasmic expression in relation to patient- and tumor characteristics in the total cohort (χ2 test for linear trend).</p

Decreased proliferation after overexpression of T-STAR in five breast cancer cell lines.

<p>A) Increased T-STAR mRNA levels after overexpression measured by RT-qPCR. B) Increased (676%) T-STAR protein level (WB) compared to GFP (16%) and wt (100%) control cells in the MDA-MB-231 cell line after overexpression. C) A decrease in proliferation could be detected at 48 h after overexpressing T-STAR using thymidine incorporation and D) after 48 h (PMC42, L56Br-C1 and T47D) or 72 h (JIMT-1 and MDA-MB-231) upon assessment of enzymatic activity (WST-1 assay). Significance is marked by a * where the p-value ≤0.05 and ** when ≤0.01.</p

Increased proliferation after knock-down of T-STAR in five breast cancer cell lines.

<p>A) Reduction in T-STAR mRNA levels after knock-down as assessed by RT-qPCR. B) Representative data showing, reduction (17%) in T-STAR protein levels (WB) compared to wt (130%) and scrambled control cells (100%) in the L56Brc1cell line after knock-down. C) Increased proliferation at 48 h (JIMT-1, MDA-MB-231 and L56Br-C1) or 72 h (PMC42 and SKBR-3) after knock-down compared to the scrambled control using thymidine incorporation (measured as cpms (counts per minutes)). Of note, values for JIMT-1 are scaled by a factor 10 for the clarity of presentation. D) An increase in proliferation was also seen measured by the WST-1 assay after 48 h (JIMT-1 and MDA-MB-231) or 72 h (PMC42, L56Br-C1 and SKBR-3). Significance is marked by a * where the p-value ≤0.05 and ** when ≤0.01.</p

Cox uni- and multivariate analysis of Recurrence Free Survival according to nuclear T-STAR expression.

<p>Note: T-STAR nuclear positive staining is defined as >10%.</p

Specification of breast cancer cell lines used in the experiments.

1<p>Kindly provided by Cecilia Hegardt, Department of Oncology, Clinical Sciences, Lund University, Skåne University Hospital.</p>2<p>Kindly provided by Paolo Cifani, Department of Immunotechnology, Lund.</p>3<p>ATCC.</p