Svetlana A. Mikheeva

Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines

ABSTRACT The expression levels of ∼4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRU infection, consistent with the G2 arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.

Dickkopf-1 mediated tumor suppression in human breast carcinoma cells.

Dickkopf-1 (DKK-1) is a secreted inhibitor of the Wnt signaling pathway. We previously identified DKK-1 as a candidate tumor suppressor and demonstrated that ectopic expression of the DKK-1 suppressed the tumorigenicity of HeLa cells in vitro and in vivo. Since suppression of tumorigenicity of HeLa cells by DKK-1 overexpression was not mediated by effects on β-catenin dependent transcription, we hypothesized that DKK-1 might also inhibit tumorigenicity of breast carcinoma cell lines lacking an activated canonical Wnt pathway. In the present study we show that ectopic expression of DKK-1 in various breast cancer cell lines resulted in a change in the cell phenotype, increased sensitivity to apoptosis, inhibition of anchorage independent growth in vitro, and suppression of tumorigenicity in vivo. Consistent with known effects of DKK-1 on the canonical Wnt signaling pathway, ectopic expression of DKK-1 in breast carcinoma cells was associated with increased phosphorylation and degradation of β-catenin. However, none of the breast tumor cells used in this study showed detectable levels of β-catenin dependent activation of TCF/Lef promoter activity measured by reporter constructs. Consistent with the results of these transient transfection assays, we were unable to demonstrate the expected β-catenin dependent, TCF/Lef mediated inhibition of cyclin D1 and c-myc gene transcription in breast cells overexpressing DKK-1. However, we found that cells with DKK-1 overexpression have increased activity of CamKII pathway. Overexpression of the constitutively active form of CamKII (T286D) resulted in inhibition of breast cancer cell tumorigenicity. Thus, our study supports the hypothesis that DKK-1 mediated tumor suppressor effect is independent of β-catenin dependent transcription and identified the CamKII pathway that contributes into DKK-1 signaling.

Periostin is a novel therapeutic target that predicts and regulates glioma malignancy.

BACKGROUND Periostin is a secreted matricellular protein critical for epithelial-mesenchymal transition and carcinoma metastasis. In glioblastoma, it is highly upregulated compared with normal brain, and existing reports indicate potential prognostic and functional importance in glioma. However, the clinical implications of periostin expression and function related to its therapeutic potential have not been fully explored. METHODS Periostin expression levels and patterns were examined in human glioma cells and tissues by quantitative real-time PCR and immunohistochemistry and correlated with glioma grade, type, recurrence, and survival. Functional assays determined the impact of altering periostin expression and function on cell invasion, migration, adhesion, and glioma stem cell activity and tumorigenicity. The prognostic and functional relevance of periostin and its associated genes were analyzed using the TCGA and REMBRANDT databases and paired recurrent glioma samples. RESULTS Periostin expression levels correlated directly with tumor grade and recurrence, and inversely with survival, in all grades of adult human glioma. Stromal deposition of periostin was detected only in grade IV gliomas. Secreted periostin promoted glioma cell invasion and adhesion, and periostin knockdown markedly impaired survival of xenografted glioma stem cells. Interactions with αvβ3 and αvβ5 integrins promoted adhesion and migration, and periostin abrogated cytotoxicity of the αvβ3/β5 specific inhibitor cilengitide. Periostin-associated gene signatures, predominated by matrix and secreted proteins, corresponded to patient prognosis and functional motifs related to increased malignancy. CONCLUSION Periostin is a robust marker of glioma malignancy and potential tumor recurrence. Abrogation of glioma stem cell tumorigenicity after periostin inhibition provides support for exploring the therapeutic impact of targeting periostin.

A syngeneic glioma model to assess the impact of neural progenitor target cell age on tumor malignancy

Human glioma incidence, malignancy, and treatment resistance are directly proportional to patient age. Cell intrinsic factors are reported to contribute to human age‐dependent glioma malignancy, but suitable animal models to examine the role of aging are lacking. Here, we developed an orthotopic syngeneic glioma model to test the hypothesis that the age of neural progenitor cells (NPCs), presumed cells of glioma origin, influences glioma malignancy. Gliomas generated from transformed donor 3‐, 12‐, and 18‐month‐old NPCs in same‐aged adult hosts formed highly invasive glial tumors that phenocopied the human disease. Survival analysis indicated increased malignancy of gliomas generated from older 12‐ and 18‐month‐old transformed NPCs compared with their 3‐month counterparts (median survival of 38.5 and 42.5 vs. 77 days, respectively). This study showed for the first time that age of target cells at the time of transformation can affect malignancy and demonstrated the feasibility of a syngeneic model using transformed NPCs for future examination of the relative impacts of age‐related cell intrinsic and cell‐extrinsic factors in glioma malignancy.

Increased age of transformed mouse neural progenitor/stem cells recapitulates age-dependent clinical features of human glioma malignancy

Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3‐ and 18‐month‐old mice into 3‐ and 20‐month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3‐month (37.5 vs. 83 days) and 20‐month (38 vs. 67 days) hosts, indicating that age‐dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF‐1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age‐dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age‐dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.

Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas

We previously identified a positive transcriptional element identical to human Ha-ras response element (HRE) within the promoter of the rat Ha-ras gene. We further identified CArG binding factor A (CBF-A), a member of heterogeneous nuclear ribonuclear protein (hnRNP) gene family, as a trans-acting factor that binds the HRE sequence with high affinity in rat mammary carcinoma cells. To determine if activation of CBF-A plays a role in tumor development in vivo, we investigated CBF-A expression and binding activity in rat mammary tumors induced by N-methyl-N-nitrosourea. We found that ~82% of tumors expressed CBF-A at levels that were 3-20 fold higher than detected in normal mammary gland. Moreover, elevated CBF-A protein levels were invariably associated with increased binding activity to the HRE. CBF-A mRNA levels in tumors were on average elevated only two fold as compared to normal mammary gland, indicating that increased CBF-A protein levels in tumors resulted from both translational and/or post-translational regulation. The level of CBF-A expression in mammary tumors was independent of Ha-ras mutational status. Together, these findings indicated that deregulation of CBF-A contributes to mammary carcinogenesis via a mechanism that is distinct from its hnRNP functions in binding and post-transcriptional regulation of RNA.

Twist1 mediated regulation of glioma tumorigenicity is dependent on mode of mouse neural progenitor transformation

Twist1 is a master regulator of epithelial mesenchymal transition and carcinoma metastasis. Twist1 has also been associated with increased malignancy of human glioma. However, the impact of inhibiting Twist1 on tumorigenicity has not been characterized in glioma models in the context of different oncogenic transformation paradigms. Here we used an orthotopic mouse glioma model of transplanted transformed neural progenitor cells (NPCs) to demonstrate the effects of Twist1 loss of function on tumorigenicity. Decreased tumorigenicity was observed after shRNA mediated Twist knockdown in HPV E6/7 Ha-RasV12 transformed NPCs and Cre mediated Twist1 deletion in Twist1 fl/fl NPCs transformed by p53 knockdown and Ha-RasV12 expression. By contrast, Twist1 deletion had no effect on tumorigenicity of NPCs transformed by co-expression of Akt and Ha-RasV12. We demonstrated a dramatic off-target effect of Twist1 deletion with constitutive Cre expression, which was completely reversed when Twist1 deletion was achieved by transient administration of recombinant Cre protein. Together these findings demonstrate that the function of Twist1 in these models is highly dependent on specific oncogenic contexts of NPC transformation. Therefore, the driver mutational context in which Twist1 functions may need to be taken into account when evaluating mechanisms of action and developing therapeutic approaches to target Twist1 in human gliomas.

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial‐to‐mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self‐renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss‐of‐function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW‐associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.