RARA Expression Research Articles

Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

BackgroundHuman peripheral blood monocytes (Mo) consist of subsets distinguished by expression of CD16 (FCγRIII) and chemokine receptors. Classical CD16- Mo express CCR2 and migrate in response to CCL2, while a minor CD16+ Mo subset expresses CD16 and CX3CR1 and migrates into tissues expressing CX3CL1. CD16+ Mo produce pro-inflammatory cytokines and are expanded in certain inflammatory conditions including sepsis and HIV infection.ResultsTo gain insight into the developmental relationship and functions of CD16+ and CD16- Mo, we examined transcriptional profiles of these Mo subsets in peripheral blood from healthy individuals. Of 16,328 expressed genes, 2,759 genes were differentially expressed and 228 and 250 were >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. CD16+ Mo were distinguished by upregulation of transcripts for dendritic cell (DC) (SIGLEC10, CD43, RARA) and macrophage (MΦ) (CSF1R/CD115, MafB, CD97, C3aR) markers together with transcripts relevant for DC-T cell interaction (CXCL16, ICAM-2, LFA-1), cell activation (LTB, TNFRSF8, LST1, IFITM1-3, HMOX1, SOD-1, WARS, MGLL), and negative regulation of the cell cycle (CDKN1C, MTSS1), whereas CD16- Mo were distinguished by upregulation of transcripts for myeloid (CD14, MNDA, TREM1, CD1d, C1qR/CD93) and granulocyte markers (FPR1, GCSFR/CD114, S100A8-9/12). Differential expression of CSF1R, CSF3R, C1QR1, C3AR1, CD1d, CD43, CXCL16, and CX3CR1 was confirmed by flow cytometry. Furthermore, increased expression of RARA and KLF2 transcripts in CD16+ Mo coincided with absence of cell surface cutaneous lymphocyte associated antigen (CLA) expression, indicating potential imprinting for non-skin homing.ConclusionThese results suggest that CD16+ and CD16- Mo originate from a common myeloid precursor, with CD16+ Mo having a more MΦ – and DC-like transcription program suggesting a more advanced stage of differentiation. Distinct transcriptional programs, together with their recruitment into tissues via different mechanisms, also suggest that CD16+ and CD16- Mo give rise to functionally distinct DC and MΦ in vivo.

Identification of genetic and epigenetic abnormalities of chromosome 3 and gene expression changes in ovarian cancer

Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers.Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines.Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARA-amplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1.The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting.

DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.

Gene Expression Profiling of Pulmonary Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Identifies New Diagnostic Markers, Biological Heterogeneity and Therapeutic Targets.

To further understand the molecular biology of mucosa-associated lymphoid tissue (MALT) lymphoma, we conducted a comprehensive gene expression study on the Affymetrix platform using total RNA extracted from biopsy specimen of 35 well-characterized pulmonary MALT lymphoma samples and compared them to gene expression profiles of a range of B and T cell lymphoma, such as Burkitt's lymphoma, diffuse large B-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, multiple myeloma, and peripheral T-cell lymphoma, and normal cellular counterparts, such as different B-cell and T-cell populations, normal plasma cells and lung tussue. Analytical strategies were adopted to negate tissue specific effect such that our results can be generalized to MALT lymphoma from other anatomical sites. We further analyzed the data using functional tools such as gene-set enrichment analysis and gene ontology. Network/pathway analysis was performed using Metacore. Unsupervised clustering of whole dataset (including other lymphoid malignancies and normal cellular counterpart) using genes with variable expression, showed that compared to other B-cell lymphoid malignancies, MALT lymphoma have a prominent T-cell signature. Using established gene expression of different B-cell compartment, we confirmed that MALT has a marginal zone B-cell and memory B-cell cell-of-origin signature. Using ANOVA with post-hoc pair-wise comparison, we identified 4 novel transcripts over-expressed in MALT but not other B-cell tumors, and validated one of these using immunohistochemistry. MALT lymphoma with t(11;18) or t(14;18) over-expressed genes enriched for pathways other than the NFKB pathway such as JAK-STAT and SRC signaling pathways, which may represent novel therapeutic targets. We identified a number of genes with ‘outlier' expression including RARA and RGS13. However, translocation involving RARA and the immunoglobulin heavy chain locus was not detected by FISH, suggesting other mechanisms of deregulating gene expression were involved. Using unsupervised clustering of only the MALT samples, we found that the molecular heterogeneity within MALT is composed of a group characterized by MALT1 translocations and another group with plasmacytic differentiation. Interestingly, further refinement of this grouping can be achieved by clustering the samples based on ‘outlier' gene expression. Samples with MALT1 translocations have high expression of RARA, samples with plasmacytic differentiation have high FKBP11 expression and samples with high RGS13 expression tend to have trisomy 3 and reactive follicles. Our study therefore identified novel molecular markers, deregulated pathways and genetic defects in MALT. Furthermore, subgroups with distinct pathological features and anchored by unique pattern of ‘outlier' gene expression were identified.

Gender differences in the predictive power of prognostic factors in NSCLC

7008 Background. In this study, we performed quantitative analysis of the gene expressions of a number of candidate prognostic factors in NSCLC and evaluated each gene expression as a prognostic factor in male and female patients (pts). Materials and Methods. Expression levels of 38 candidate prognostic genes were determined by quantitative real time RT-PCR (Taqman®) in 91 tumor tissues from pts with curatively resected NSCLC stage I to IIIa. This group included 21 female pts. All tumors were R0 resected. Pts with stage IIIa received adjuvant radiotherapy. Statistical methods were used to find gene expression cut-off values that separated pts into groups with different survival. Results.Overall median survival (MS) was was not significantly different for males and females (51 vs 44 months, respectively). Females had significantly higher median expression of Bax, Her2, COX-1, RARa, RXRb and RXRg than males, whereas median EGFR was higher among males than females. Gene expressions of Her2, ERCC1, RXRb were female-specific prognostic factors. For female pts with high Her2 expression (>13) MS was not reached, but for low Her2 (<13), MS was 26 months (p=0.006). High ERCC1 (> 12) gave MS of 60 months, but 29 months for low ERCC1 (<13) (p=0.026). For high RXRb (>9) MS was 59 months but 20 months for LOW RXRb (< 9) (p=0.019). Differential survival of males was not significantly predicted by these gene expressions. However, high COX-2 expression (>1) significantly predicted for shorter survival in males (p=0.028) but not in females and low ODC (<11) was a significant predictor of shorter survival in males (p=0.008) but not in females. Conclusion. Our data show that prognostic factors can be strongly gender-specific and that a marker that is not useful for one sex can be highly predictive in the other. Pt groups should be separated by genders to derive maximal benefit from prognostic factors. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics, Inc. Response Genetics, Inc. Response Genetics, Inc.

Gender differences in the predictive power of prognostic factors in NSCLC

7008 Background. In this study, we performed quantitative analysis of the gene expressions of a number of candidate prognostic factors in NSCLC and evaluated each gene expression as a prognostic factor in male and female patients (pts). Materials and Methods. Expression levels of 38 candidate prognostic genes were determined by quantitative real time RT-PCR (Taqman®) in 91 tumor tissues from pts with curatively resected NSCLC stage I to IIIa. This group included 21 female pts. All tumors were R0 resected. Pts with stage IIIa received adjuvant radiotherapy. Statistical methods were used to find gene expression cut-off values that separated pts into groups with different survival. Results.Overall median survival (MS) was was not significantly different for males and females (51 vs 44 months, respectively). Females had significantly higher median expression of Bax, Her2, COX-1, RARa, RXRb and RXRg than males, whereas median EGFR was higher among males than females. Gene expressions of Her2, ERCC1, RXRb were female-specific prognostic factors. For female pts with high Her2 expression (>13) MS was not reached, but for low Her2 (<13), MS was 26 months (p=0.006). High ERCC1 (> 12) gave MS of 60 months, but 29 months for low ERCC1 (<13) (p=0.026). For high RXRb (>9) MS was 59 months but 20 months for LOW RXRb (< 9) (p=0.019). Differential survival of males was not significantly predicted by these gene expressions. However, high COX-2 expression (>1) significantly predicted for shorter survival in males (p=0.028) but not in females and low ODC (<11) was a significant predictor of shorter survival in males (p=0.008) but not in females. Conclusion. Our data show that prognostic factors can be strongly gender-specific and that a marker that is not useful for one sex can be highly predictive in the other. Pt groups should be separated by genders to derive maximal benefit from prognostic factors. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics, Inc. Response Genetics, Inc. Response Genetics, Inc.

Expression of Retinoic Acid Receptor Alpha mRNA in Human Leukemia Cells

AbstractThe expression of the newly described human retinoic acid receptor alpha (RARα) in six nonlymphoid and six lymphoid leukemia cell lines and nine freshly obtained samples of leukemia cells from patients with acute nonlymphoid leukemia was assessed by Northern blot analysis, using a full length cDNA clone of RARα as probe. RARa was expressed in all 12 cell lines and in all fresh leukemia samples as two major transcripts of 2.6 and 3.5 kb in size. Levels of RARa expression and transcript sizes in retinoid-sensitive cells (such as HL60 or fresh promyelocytic leukemia cells) were not different from those in other samples. Moreover, expression of RARα was not significantly modulated by exposure to cis-retinoic acid (cisRA) in either cisRA- responsive or unresponsive cells. By using a 3' fragment of the RARα gene as a probe, we confirmed that the transcripts visualized did not represent the homologous RARβ gene. RARα appears to be expressed in most human leukemia cells regardless of the type of biologic response to retinoic acid.