Q&A Meet the Author: Hector L. Franco
Hector L. Franco. Cell Genomics. 2025 Feb 12;5(2):100772.
Hector Franco is a group leader at the University of Puerto Rico Comprehensive Cancer Center (UPRCCC), having re-located from the University of North Carolina in 2023. His laboratory focuses on gene regulation, non-coding RNA, and the tumor microenvironment. In this issue of Cell Genomics, his team presents the resource “Defining the regulatory logic of breast cancer using single-cell epigenetic and transcriptome profiling,” which highlights non-coding mechanisms of gene regulation in breast cancer.
Defining the Regulatory Logic of Breast Cancer Using Single-Cell Epigenetic and Transcriptome Profiling
Matthew J. Regner, Susana Garcia-Recio, Aatish Thennavan, Kamila Wisniewska, Raul Mendez-Giraldez, Brooke Felsheim, Philip M. Spanheimer, Joel S. Parker, Charles M. Perou, and Hector L. Franco. Cell Genomics. 2025
Annotation of cis-regulatory elements that drive transcriptional dysregulation in cancer cells is critical to understanding tumor biology. Herein, we present matched chromatin accessibility (single-cell assay for transposase-accessible chromatin by sequencing [scATAC-seq]) and transcriptome (single-cell RNA sequencing [scRNA-seq]) profiles at single-cell resolution from human breast tumors and healthy mammary tissues processed immediately following surgical resection. We identify the most likely cell of origin for subtype-specific breast tumors and implement linear mixed-effects modeling to quantify associations between regulatory elements and gene expression in malignant versus normal cells. These data unveil cancer-specific regulatory elements and putative silencer-to-enhancer switching events in cells that lead to the upregulation of clinically relevant oncogenes. In addition, we generate matched scATAC-seq and scRNA-seq profiles for breast cancer cell lines, revealing a conserved oncogenic gene expression program between in vitro and in vivo cells. This work highlights the importance of non-coding regulatory mechanisms that underlie oncogenic processes and the ability of single-cell multi-omics to define the regulatory logic of cancer cells.
CRISPR-Enabled Screening of Transcribed Super-Enhancers in Triple-Negative Breast Cancer Identifies Drivers of Tumor Progression
Michael W. Lewis, Caitlin M. King, Kamila Wisniewska,
Matthew J. Regner, Alisha Coffey, Michael R. Kelly,
Raul Mendez-Giraldez, Eric S. Davis, Douglas H. Phanstiel,
and Hector L. Franco. Cancer Research . 2024
Triple-negative breast cancer (TNBC) is the most therapeutically
recalcitrant form of breast cancer, which is due in part to the paucity
of targeted therapies. A systematic analysis of regulatory elements
that extend beyond protein-coding genes could uncover avenues
for therapeutic intervention. To this end, we analyzed the regulatory
mechanisms of TNBC-specific transcriptional enhancers together with
their noncoding enhancer RNA (eRNA) transcripts. The functions of
the top 30 eRNA-producing super-enhancers were systematically
probed using high-throughput CRISPR-interference assays coupled
to RNA sequencing that enabled unbiased detection of target genes
genome-wide. Generation of high-resolution Hi-C chromatin interaction
maps enabled annotation of the direct target genes for each super-enhancer,
which highlighted their proclivity for genes that portend worse clinical
outcomes in patients with TNBC. Illustrating the utility of this dataset,
deletion of an identified super-enhancer controlling the nearby PODXL
gene or specific degradation of its eRNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared with nonmalignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of TNBC.
Tamoxifen Response at Single-Cell Resolution in Estrogen Receptor-Positive Primary Human Breast Tumors
Hyunsoo Kim, Austin A. Whitman, Kamila Wisniewska, Rasha T. Kakati, Susana Garcia Recio, Benjamin C. Calhoun, Hector L. Franco, Charles M. Perou, and Philip M. Spanheimer. 2023. Clinical Cancer Res. 2023
Single-Cell Transcriptional and Epigenetic Profiles of Male Breast Cancer Nominate Salient Cancer-Specific Enhancers
Hyunsoo Kim, Kamila Wisniewska, Matthew J. Regner, Aatish Thennavan, Philip M. Spanheimer and Hector L. Franco. Int. J. Mol Sci. 2023
A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer
Michael R. Kelly, Kamila Wisniewska, Matthew J. Regner, Michael W. Lewis, Andrea A. Perreault, Eric S. Davis, Douglas H. Phanstiel, Joel S. Parker, Hector L. Franco. Nature Communications 2022.
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The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we utilize an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We systematically probe the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we construct chromatin interaction maps that enable the annotation of direct target genes for these super-enhancers and confirm their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach examines a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.
Enhancer RNA Transcription is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer
Michael W. Lewis, Kamila Wisniewska, Caitlin M. King, Shen Li, Alisha Coffey, Michael R. Kelly, Matthew J. Regner, Hector L. Franco. Cancers. 2022
Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via
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CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention.
A multi-omic single-cell atlas of human gynecologic malignancies
Matthew Regner, Kamila Wisniewska, Susana Garcia-Recio, Aatish Thennavan, Raul Mendez-Giraldez, Venkat S. Malladi, Gabrielle Hawkins, Joel S. Parker, Charles M. Perou, Victoria L. Bae-Jump, Hector L. Franco. Molecular Cell. 2021. https://doi.org/10.1016/j.molcel.2021.10.013
Deconvolution of regulatory mechanisms that drive transcriptional programs in cancer cells is key to understanding tumor biology. Herein, we present matched transcriptome (scRNA-seq) and chromatin accessibility profiles (scATAC-seq) at single-cell resolution from human ovarian and endometrial tumors processed immediately following surgical resection. This dataset reveals the complex heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression. We show that malignant cells acquire previously unannotated regulatory elements
to drive hallmark cancer pathways. Moreover, malignant cells from within the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intratumoral heterogeneity. Finally, we infer the malignant cell type-specific activity of transcription factors. By defining the regulatory logic of cancer cells, this work reveals an important reliance on oncogenic regulatory elements and highlights the ability of matched scRNAseq/scATACseq to uncover clinically relevant mechanisms of tumorigenesis in gynecologic cancers.
Shen Li, Joseph P. Garay, Colby A. Tubbs, Hector L. Franco. FEBS Open Bio. 2021. https://doi.org/10.1002/2211-5463.13139
Total Functional Score of Enhancer Elements Identifies Lineage-Specific Enhancers that Drive Differentiation of Pancreatic Cells
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​Venkat Malladi, Anusha Nagari, Hector Franco, W. Lee Kraus. Bioinformatics and Biology Insights. 2020 Jul 1; 14-20.
Transcriptional Control by Enhancers and Enhancer RNAs
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Michael W. Lewis, Shen Li, Hector L. Franco. Transcription. 2019. https://doi.org/10.1080/21541264.2019.1695492
The regulation of gene expression is a fundamental cellular process and its misregulation is a key component of disease. Enhancers are one of the most salient regulatory elements in the genome and help orchestrate proper spatiotemporal gene expression during development, in homeosta- sis, and in response to signaling. Notably, molecular aberrations at enhancers, such as transloca- tions and single nucleotide polymorphisms, are emerging as an important source of human variation and susceptibility to disease. Herein we discuss emerging paradigms addressing how genes are regulated by enhancers, common features of active enhancers, and how non-coding enhancer RNAs (eRNAs) can direct gene expression programs that underlie cellular phenotypes. We survey the current evidence, which suggests that eRNAs can bind to transcription factors, mediate enhancer-promoter interactions, influence RNA Pol II elongation, and act as decoys for repressive cofactors. Furthermore, we discuss current methodologies for the identification of eRNAs and novel approaches to elucidate their functions.
Enhancer Transcription Reveals Subtype-Specific
Gene Expression Programs Controlling Breast Cancer Pathogenesis
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Hector L. Franco, Anusha Nagari, Venkat Malladi, Wenqian Li, Yuanxin Xi, Dana Richardson, Kaori Tanaka, Jing Li, Michelle C. Barton, Xiaobing Shi, Khandan Keyomarsi, Mark T. Bedford, Wei Li, Sharon Y. R. Dent, W. Lee Kraus. Genome Research. 2018. 28: 159-170
Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer
cell lines representing five major molecular subtypes of breast cancer, as well as two immortal- ized (“normal”) human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of en- hancer transcription, TF mRNA expression levels, TF motif P-values, and enrichment of H3K4me1 and H3K27ac. When ap- plied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.
Histone Modification Profiling in Breast Cancer Cell Lines Highlights Commonalities and Differences Among Subtypes
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Yuanxin Xi, Wenqian Li, Kaori Tanaka, Kendra L. Allton, Dana Richardson, Jing Li, Hector L. Franco, Anusha Nagari, Venkat Malladi, Khandan Keyomarsi, Jianjun Shen, Mark T. Bedford, Xiaobing Shi, Michelle C. Barton, W. Lee Kraus, Wei Li, Sharon Y. R. Dent. BMC Genomics. 2018. 19:150
No Driver Behind the Wheel? Targeting Transcription as a Therapeutic Strategy in Cancer
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Hector L. Franco, W. Lee Kraus. Cell. 2015. 163(1):28-30.
TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites to Alter the Breast Cancer Cell Transcriptome
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Hector L. Franco, Anusha Nagari, W. Lee Kraus. Molecular Cell. 2015. 2;58(1):21-34.
The interplay between mitogenic and proinflamma- tory signaling pathways plays key roles in deter- mining the phenotypes and clinical outcomes of breast cancers. Using GRO-seq in MCF-7 cells, we defined the immediate transcriptional effects of crosstalk between estradiol (E2) and TNFa, identi- fying a large set of target genes whose expression is rapidly altered with combined E2 + TNFa treat- ment, but not with either agent alone. The pleiotropic effects on gene transcription in response to E2 + TNFa are orchestrated by extensive remodeling of the ERa enhancer landscape in an NF-kB- and FoxA1-dependent manner. In addition, expression of the de novo and synergistically regulated genes is strongly associated with clinical outcomes in breast cancers. Together, our genomic and molecu- lar analyses indicate that TNFa signaling, acting in pathways culminating in the redistribution of NF-kB and FoxA1 binding sites across the genome, creates latent ERa binding sites that underlie altered patterns of gene expression and clinically relevant cellular responses.
Nonsense mutations of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients cause dysregulation of periostin.
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Hector L. Franco, Jose J. Casasnovas, Ruth G. Leon, Robert Friesel, Yongchao Ge, Robert J. Desnick, Carmen L. Cadilla. Int Journal Biochemistry and Cell Biology. 2011. 43(10):1523-31
Homozygous nonsense mutations in TWIST2 cause Setleis syndrome.
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​Turgut Tukel, Drazen Šošić, Lidah I. Al-Gazali, Mónica Erazo, Jose Casasnovas, Hector L. Franco, James A. Richardson, Eric N. Olson,2 Carmen L. Cadilla, Robert J. Desnick. American Journal of Human Genetics. 2010. 13;87(2):289-96