Princy Francis

A p21-ZEB1 Complex Inhibits Epithelial-Mesenchymal Transition through the MicroRNA 183-96-182 Cluster

ABSTRACT The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21+/+ and p21−/− cells. Three abundant miRNA clusters, miR-200b-200a-429, miR-200c-141, and miR-183-96-182, were downregulated in p21-deficient cells. Consistent with the known function of the miR-200 family and p21 in inhibition of the epithelial-mesenchymal transition (EMT), we observed EMT upon loss of p21 in multiple model systems. To explore a role of the miR-183-96-182 cluster in EMT, we identified its genome-wide targets and found that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1, and KLF4. Reintroduction of miR-200, miR-183, or miR-96 in p21−/− cells inhibited EMT, cell migration, and invasion. Conversely, antagonizing miR-200 and miR-183-96-182 cluster miRNAs in p21+/+ cells increased invasion and elevated the levels of VIM, ZEB1, and SLUG mRNAs. Furthermore, we found that p21 forms a complex with ZEB1 at the miR-183-96-182 cluster promoter to inhibit transcriptional repression of this cluster by ZEB1, suggesting a reciprocal feedback loop.

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

Significance In the colon, stem cell self-renewal and multipotency is regulated by the polycomb complex protein BMI1, among other genes. Differentiation is regulated by the transcription factor caudal-type homeobox 1 (CDX1), expression of which coincides with repression of BMI1. Colorectal cancer stem cells (CSCs) express BMI1 but not CDX1. Tumors that silence CDX1 have a higher proportion of CSCs and an undifferentiated histology, whereas aberrant CDX1 expression is associated with intestinal metaplasias such as Barretts esophagus. We have identified microRNA-215 (miR-215) as a target of CDX1 in colon cancer that mediates repression of BMI1. MiR-215 operates downstream of CDX1 to promote differentiation and inhibit stemness. In combination with recent advances in the therapeutic uses of small RNAs, miR-215 could offer a novel method to specifically target CSCs. The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP–PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.

Enforced expression of Lin28b leads to impaired T-cell development, release of inflammatory cytokines, and peripheral T-cell lymphoma

LIN28A and LIN28B, the mammalian homologs of lin-28, are implicated in malignant transformation in part because of their ability to promote degradation of the let-7 family of miRs. In the present study, we show that overexpression of Lin28b in vivo leads to an aggressive peripheral T-cell lymphoma (PTCL) characterized by widespread infiltration of parenchymal organs with malignant CD4(+) cells. Similar to patients with PTCL, Lin28b-transgenic mice show signs of inflammation such as eosinophilia, increased C-reactive protein, release of inflammatory cytokines, and pleural effusion. The PTCLs that develop in Lin28b mice are derived from activated T cells and show decreased let-7 expression, increased Il6 expression, activation of NF-κB, and infiltration of B cells, all resulting in an inflammatory microenvironment. In addition, LIN28B is overexpressed 7.5-fold in PTCL patient samples compared with activated CD4(+) cells. The results of the present study demonstrate for the first time that Lin28b can transform primary cells in vivo, identify a previously unsuspected link between Lin28b and PTCL, and provide a unique animal model for the study of PTCL biology and therapy.

miR-23a impairs bone differentiation in osteosarcoma via down-regulation of GJA1

Osteosarcoma is the most common type of bone cancer in children and adolescents. Impaired differentiation of osteoblast cells is a distinguishing feature of this aggressive disease. As improvements in survival outcomes have largely plateaued, better understanding of the bone differentiation program may provide new treatment approaches. The miRNA cluster miR-23a~27a~24-2, particularly miR-23a, has been shown to interact with genes important for bone development. However, global changes in gene expression associated with functional gain of this cluster have not been fully explored. To better understand the relationship between miR-23a expression and bone cell differentiation, we carried out a large-scale gene expression analysis in HOS cells. Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system. Downstream bioinformatic analysis identified miR-23a target gene connexin-43 (Cx43/GJA1), a mediator of intercellular signaling critical to osteoblast development, as acutely affected by miR-23a levels. Connexin-43 is up-regulated in the course of HOS cell differentiation and is down-regulated in cells transfected with miR-23a. Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation. Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay. This work demonstrates novel interactions between microRNA expression, intercellular signaling and bone differentiation in osteosarcoma.

Abstract 4861: Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes

Background: Next generation sequencing (NGS) has revealed that the genetic profiles of individual tumors are highly heterogeneous due to their subclonal composition. Tumor heterogeneity has profound clinical implications affecting differences in treatment response and therapeutic resistance. Methods: An estrogen receptor positive, Her2 normal breast cancer patient underwent definitive surgical treatment with modified radical mastectomy. Multiple samples were obtained from the primary cancer and metastatic lymph nodes. Flow cytometry based methods were used to isolate and classify distinct cell subpopulations according to their ploidy, and samples were processed for whole exome sequencing (WES). Comparative Genomic Hybridization (CGH) methods were used to identify somatic unique copy-number alterations (CNAs). WES-Single Nucleotide Variations (SNVs) were used to infer subclonal phylogenetic relationships using Maximum Parsimony methods and annotated with variation-cluster-barcodes (vc-barcodes) generated using a variation Bayesian mixture model (VBMM) approach that focuses on copy-number neutral sequence variations for subclone identification (SciClone). Our comparative phylogenetic-VBMM clustering method was used to identify CNA and/or SNV drivers that are key in differentiating subclonal populations at multiple tumor sites. Results: CNA-neutral WES-SNVs from 25 subclonal populations (isolated from 4 distinct sites) clustered into 6 major groups and were used to generate vc-barcodes. A phylogenetic reconstruction of subclonal architecture annotated with vc-barcode information expedited identification of key variations underlying the differentiation of subclones at distinct tumor sites. For example, we identified an amplification event involving the Anaplastic Lymphoma Kinase (ALK) gene that was more frequent in primary biopsy subclones (12/17, ∼71%) in comparison to metastatic subclones (2/6, ∼34%). As the ALK amplification is lost, we find that most of the metastatic subclones also acquire a predicted damaging mutation in this oncogene (4/6, ∼67%). In addition, we identified a potential driver metastatic cell lineage that carries the ALK amplification in the absence of the nonsynonymous mutation. Conclusions: We developed a hybrid clustering methodology and used it to reconstruct the subclonal architecture in primary and metastatic tumors from a single breast cancer patient. Our methods have identified several CNAs and/or SNVs underlying subclonal differentiation, as well as potential biomarkers of disease progression and indicators of emerging resistance. Application of these methods to larger cohorts and types of tumors should be conducted to ascertain more precise estimates of the predictive accuracy of our processes. Citation Format: Mia D. Champion, Princy Francis, Barbara A. Pockaj, Michael T. Barrett. Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes. [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 4861. doi:10.1158/1538-7445.AM2015-4861

Abstract 3147: Cancer-specific mutations in adult soft tissue sarcomas.

Understanding of how cancer initiates and progresses is a fundamental goal of cancer research. Identification of cancer mutations is of profound importance for both understanding of the disease and for developing novel therapies. Soft-tissue sarcomas include multiple uncommon histotypes with features of diverse connective tissue lineages. Although some of their underlying genetic changes have been elucidated, for example in those types which contain etiologic chromosome translocations, in general, the genetic basis of these tumors is incompletely described. To investigate mutations in 8 subtypes of adult soft tissue sarcomas, we resequenced coding exons from about 1300 cancer relevant genes in 66 tumor samples. Data was analyzed for somatic variants in samples with matched normals while samples without normal were analyzed individually. The most frequent events were TP53 mutations (26%) followed by RB1 (9%) and APC (8%) mutations. Even though, well-defined oncogene activating mutations were present, they were very rare. Given the prevalence of tumor suppressor mutations in these samples we are currently integrating CGH results to look for chromosomal deletions to support loss of tumor suppressor genes. In addition, we are investigating low-frequency mutations in lesser-known genes that may be of potential interest. Our preliminary results suggest that these subtypes of soft-tissue sarcomas depend on tumor-suppressor loss predominantly, and may have gained multiple complex sets of mutations in a number of other pathways. In conclusion, our work will be important in cataloging novel cancer mutations in rare types of adult sarcomas, with potential implications for understanding their biology and the development of new therapies. Citation Format: Ogan D. Abaan, Princy Francis, Marbin Pineda, Robert L. Walker, Yuelin J. Zhu, Sven Bilke, Sean R. Davis, Paul S. Meltzer. Cancer-specific mutations in adult soft tissue sarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2013-3147

Abstract 5331: A novel function of p21 in inhibition of epithelial-mesenchymal transition through microRNAs.

The tumor suppressor p21 inhibits cell proliferation during the stress response. However, p21 can also directly regulate gene expression by repressing specific transcription factors. Here, we identified p21-regulated miRNAs by sequencing small RNAs from HCT116-p21+/+ and HCT116-p21-/- cells. Three abundant clusters, miR-200b-200a-429, miR-200c-141 and miR-183-96-182 were down-regulated in p21-depleted HCT116 and MCF10A cells. Loss of p21 induced epithelial-mesenchymal transition (EMT) and enhanced migration and invasion in multiple model systems. Identification of genome-wide targets of the miR-183-96-182 cluster indicated that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1 and KLF4, to inhibit EMT, migration and invasion. In turn, elevated ZEB1 levels in HCT116-p21-/- cells directly repressed miR-183-96-182 cluster transcription, revealing a feedback loop. Re-introduction of miR-200, miR-183 or miR-96 in HCT116-p21-/- cells inhibited migration and invasion. These novel findings suggest that coordinated down-regulation of three miRNA clusters upon loss of p21 in unstressed cells promotes EMT, migration and invasion. Citation Format: Xiao L. Li, Toshifumi Hara, Youngeun Choi, Murugan Subramanian, Princy Francis, Sven Bilke, Robert L. Walker, Marbin Pineda, Yu-an Yang, Ji Luo, Lalage M. Wakefield, Ben H. Park, Thomas Brabletz, Dipanjan Chowdhury, Paul S. Meltzer, Ashish Lal. A novel function of p21 in inhibition of epithelial-mesenchymal transition through microRNAs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5331. doi:10.1158/1538-7445.AM2013-5331

Abstract 1113: Role of the microRNA-23∼27∼24 clusters in osteosarcoma

Osteosarcomas are rare but aggressive tumors that predominantly affect adolescents. These neoplasms of mesenchymal origin are biologically heterogeneous and produce immature osteoid or bone. The pathogenesis of osteosarcomas is poorly understood although defective osteogenic differentiation seems to play a key role. In order to elucidate the underlying molecular mechanisms, 16 osteosarcoma cell lines, 2 osteoblast cell lines and 45 osteosarcoma tumors were studied using a combination of high-throughput techniques such as microRNA (miRNA) and gene expression profiling, miRNA sequencing and array comparative genomic hybridization. Overexpression of the miRNA clusters miR-23a∼27a∼24-2 (19p13.13) and miR-23b∼27b∼24-1 (9q22.32) was identified in a subset of the osteosarcoma tumors and cell lines. Furthermore, quantitative RT-PCR revealed that the miR-23a and miR-23b clusters were downregulated during osteoblast differentiation of the osteosarcoma cell line HOS and overexpression of these miRNAs (by transient transfection of miRNA mimics) delayed osteoblast differentiation and matrix mineralization evaluated by alkaline phosphatase and Alizarin Red staining. Target gene identification experiments were then performed for each of the members of the miR-23a and miR-23b clusters. Gain of function (using miRNA mimics for exogenous expression) and loss of function (using antisense miRNAs to inhibit endogenous expression) experiments followed by downstream gene expression analysis identified several putative targets in osteosarcoma cell lines HOS and HuO3N1. The gene lists were highly enriched in targets (including differentiation genes) identified by TargetScan and other target prediction software. These results suggest that deregulation of the miR-23∼27∼24 clusters may play a role in defective osteogenic differentiation and osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1113. doi:1538-7445.AM2012-1113

Abstract 3934: Identification of novel cancer DNA sequence variants in human sarcomas

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Understanding of the genetic basis of cancer initiation and progression is a fundamental goal of cancer research. Identification of cancer variants is of profound importance for both understanding disease mechanism and for developing novel therapies. Soft-tissue sarcomas are multiple uncommon histotypes with features of diverse connective tissue lineages. Although some of their underlying genetic changes have been elucidated, for example in those types which contain etiologic chromosome translocations, in general, the genetics of these tumors is incompletely described. Adult soft tissue sarcomas often exhibit highly complex genomic rearrangements. To search for novel variants in these tumors, we utilized a custom “targeted-resequencing” approach to sequence coding exons from over 1300 known cancer-related genes and candidate gene families in 65 soft-tissue sarcoma patient samples covering several subtypes and their matching normal tissues where available. We used the previously published method of “In-solution capture” to enrich for regions of interest and sequenced both the normals and the tumors using the Illumina GAII platform. The variant calls reveal the distribution of mutations affecting the exons of the targeted regions across histotypes, and reveal the role of critical functional pathways in sarcoma genesis. Preliminary data suggest that TP53 single nucleotide variants are mostly confined to one histotype. This work is an important step in cataloging novel cancer mutations in these cancers, with potential implications for understanding their biology and the development of new therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2011-3934

Abstract 2213: Identification of novel cancer mutations in sarcomas

Understanding of how cancer initiates and progresses is the fundamental of cancer research. Identification of cancer mutations is of profound importance for both understanding of the disease and for developing novel therapies. To search novel cancer mutations, we have utilized a “targeted resequencing” approach to resequence coding exons from about 1300 genes in a panel of various sarcoma patient samples. We used the previously published method of “In-solution capture” to enrich for regions of interest and resequenced the enriched population on an Illumina GAII platform using single-ended or paired ended reads. The performance and depth of the enrichment and resequencing was adequate for variant calling. We obtained roughly 70% on genome reads and about 30% of the reads fall on target while about 40% of the reads fall on target±500bp. An in house variant calling script was used for SNP/variant calling. Each sample yielded multiple genes with novel variations some of which are either missense or nonsense and in a highly conserved residue that were also previously identified in other cancers as well. Preliminary results from samples resequenced so far demonstrate that these sarcoma samples have variations in a variety of genes with diverse functions, including but not limited to signaling pathway intermediates. In conclusion, our work will be important in cataloging novel cancer mutations in rare sarcomas, with potential implications for understanding their biology and the development of new therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2213.