Low E2F2 activity is associated with high genomic instability and PARPi resistance

AbstractThe E2F family, classically known for a central role in cell cycle, has a number of emerging roles in cancer including angiogenesis, metabolic reprogramming, metastasis and DNA repair. E2F1 specifically has been shown to be a critical mediator of DNA repair; however, little is known about DNA repair and other E2F family members. Here we present an integrative bioinformatic and high throughput drug screening study to define the role of E2F2 in maintaining genomic integrity in breast cancer. We utilized in vitro E2F2 ChIP-chip and over expression data to identify transcriptional targets of E2F2. This data was integrated with gene expression from E2F2 knockout tumors in an MMTV-Neu background. Finally, this data was compared to human datasets to identify conserved roles of E2F2 in human breast cancer through the TCGA breast cancer, Cancer Cell Line Encyclopedia, and CancerRx datasets. Through these methods we predict that E2F2 transcriptionally regulates mediators of DNA repair. Our gene expression data supports this hypothesis and low E2F2 activity is associated with a highly unstable tumor. In human breast cancer E2F2, status was also correlated with a patient’s response to PARP inhibition therapy. Taken together this manuscript defines a novel role of E2F2 in cancer progression beyond cell cycle and could impact patient treatment.</jats:p

Low E2F2 activity is associated with high genomic instability and PARPi resistance

AbstractThe E2F family, classically known for a central role in cell cycle, has a number of emerging roles in cancer including angiogenesis, metabolic reprogramming, metastasis and DNA repair. E2F1 specifically has been shown to be a critical mediator of DNA repair; however, little is known about DNA repair and other E2F family members. Here we present an integrative bioinformatic and high throughput drug screening study to define the role of E2F2 in maintaining genomic integrity in breast cancer. We utilizedin vitroE2F2 ChIP-chip and over expression data to identify transcriptional targets of E2F2. This data was integrated with gene expression from E2F2 knockout tumors in an MMTV-Neu background. Finally, this data was compared to human datasets to identify conserved roles of E2F2 in human breast cancer through the TCGA breast cancer, Cancer Cell Line Encyclopedia, and CancerRx datasets. Here we have computationally predicted that E2F2 transcriptionally regulates key mediators of DNA repair. Our gene expression data supports this hypothesis and low E2F2 activity is associated with a highly unstable tumor. In human breast cancer E2F2, status was also correlated with a patient’s response to PARP inhibition therapy. Taken together this manuscript defines a novel role of E2F2 in cancer progression beyond cell cycle and could be therapeutically relevant.Author SummaryThe E2F family of proteins have been known to regulate cell cycle and have recently been shown to have a number of roles in tumor progression. Here we use a combination of computational techniques and high-throughput drug screening data to establish a novel role of E2F2 in maintaining genomic integrity. We have shown that a number of direct and indirect target genes of E2F2 are involved in multiple classical DNA repair pathways. Importantly, this was shown to be unique to E2F2 and not present with other activator E2Fs like E2F1. We have also shown that E2F2 activity is positively correlated with PARP inhibitor sensitivity regardless of BRCA1/2 status. This is important due to the recent approval of PARP inhibitor therapy in the clinic. Based on our work E2F2 activity could serve as a novel biomarker of response and may identify a new cohort of patients which could benefit from PARPi therapy.</jats:sec

Abstract 488: E2F2-mediated copy number changes drive metastasis and therapeutic response of HER2-positive tumors through Col1a1, CHAD, and AKT-dependent mechanisms

Abstract The E2F family of transcription factors is classically known to regulate G1 to S-phase transition in cell cycle but has emerging roles in HER2+ breast cancer. A loss of E2F1 or E2F2, in a HER2 mouse model, MMTV-Neu, leads to a decrease in tumor metastasis1. It is not known what mechanistic roles specific E2Fs are playing in this process. To investigate this, we leveraged and bioinformatic principles, including genomics and transcriptomics with traditional laboratory science and high throughput drug screening projects. This experimental approach immediately revealed that loss of E2F2 is significantly associated with a more unstable tumor including an increase in copy number alterations, single nucleotide variants, and translocations. Further analysis revealed a conserved copy number alteration in both mouse and humans. Specifically we noted the amplification of 17q21.33 in 25% of HER2+ patients. The analogous region chromosome 11D was lost in 30% of the less metastatic MMTV-Neu E2F2 knockout mice indicating a role of the region in tumor metastasis. Transcriptomic data revealed that two genes, Collagen Type I, alpha 1 (Col1a1) and Chondroadherin (CHAD), were potential genes of significance in the amplification event. CRISPR mediated knockout studies were conducted to determine each gene’s effect on tumor cell migration and metastasis in mouse and human derived cell lines. Wound healing assays and tail vein injection have shown the Col1a1 and CHAD KO cell lines have a delay in cell migration (P&amp;lt;.01) and reduced ability to colonize the lung (P&amp;lt;.05) respectively. Oncogenic signaling data shows that the 17q21.33 amplification event has higher AKT and E2F2 signaling than HER2 positive tumors without the event. It was hypothesized that the tumors would be dependent on the signaling and perturbation of the network might be an effective therapy for patients with the 17q21.33 amplification event. To investigate this we identified deferentially lethal siRNAs and compounds between HER2+ tumors with and without the 17q21.33 event in the Achilles, CCLE, and PDX datasets. A String-DB analysis showed many of the deferentially lethal genes and compounds centered around AKT. A decrease in AKT signaling through siRNA or chemical compound results in the death of the cell in the 17q21.33 amplified samples but not the HER2+ samples without the event. This study reveals that patients’ with a 17q21.33 amplification event have more metastatic tumors mediated through Col1a1 and CHAD and may be responsive to AKT targeting therapy. References1. Andrechek ER: HER2/Neu tumorigenesis and metastasis is regulated by E2F activator transcription factors. Oncogene 2013. Citation Format: Jonathan P. Rennhack, Kelian Sun, Jordan Honeysett, Eran Andrechek. E2F2-mediated copy number changes drive metastasis and therapeutic response of HER2-positive tumors through Col1a1, CHAD, and AKT-dependent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2017-488</jats:p

Elucidating Genes that Promote the Development of Stem-Like Cells in Triple Negative Breast Cancers

Faculty advisor: Dr. Justin HwangThis research was supported by the Undergraduate Research Opportunities Program (UROP).Makovec, Allison; Tape, Sydney; Richter, Camden; Rennhack, Jonathan P.; Hwang, Justin. (2022). Elucidating Genes that Promote the Development of Stem-Like Cells in Triple Negative Breast Cancers. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/250070

From proposal to poster: course-based undergraduate research experience in a physiology laboratory course

The laboratory course is an excellent venue to apply content, practice inquiry, improve critical thinking, practice key clinical skills, and work with data. The use of inquiry-based course projects allows for students to propose open ended questions, form a hypothesis, design an experiment, collect data, analyze data, draw conclusion, and present their findings. This comprehensive experience is ideal for a capstone (senior level) laboratory course that is the culmination of 4 yr of study in the degree. At Michigan State University, the capstone laboratory has incorporated a formal course-based research experience in human physiology. The rationale and logistics for running such an experience are described in this paper. </jats:p

Genomic Amplification and Functional Dependency of the Gamma Actin Gene ACTG1 in Uterine Cancer

Sarcomere and cytoskeleton genes, or actomyosin genes, regulate cell biology including mechanical stress, cell motility, and cell division. While actomyosin genes are recurrently dysregulated in cancers, their oncogenic roles have not been examined in a lineage-specific fashion. In this report, we investigated dysregulation of nine sarcomeric and cytoskeletal genes across 20 cancer lineages. We found that uterine cancers harbored the highest frequencies of amplification and overexpression of the gamma actin gene, ACTG1. Each of the four subtypes of uterine cancers, mixed endometrial carcinomas, serous carcinomas, endometroid carcinomas, and carcinosarcomas harbored between 5~20% of ACTG1 gene amplification or overexpression. Clinically, patients with ACTG1 gains had a poor prognosis. ACTG1 gains showed transcriptional patterns that reflect activation of oncogenic signals, repressed response to innate immunity, or immunotherapy. Functionally, the CRISPR-CAS9 gene deletion of ACTG1 had the most robust and consistent effects in uterine cancer cells relative to 20 other lineages. Overall, we propose that ACTG1 regulates the fitness of uterine cancer cells by modulating cell-intrinsic properties and the tumor microenvironment. In summary, the ACTG1 functions relative to other actomyosin genes support the notion that it is a potential biomarker and a target gene in uterine cancer precision therapies

Abstract 2402: SMAD4 represses PDAC metastatic colonization through binding FOSL1 enhancer regions

Abstract Less than 5% of patients diagnosed with pancreatic ductal adenocarcinomas (PDAC) survive more than 5 years, largely due to therapeutic resistance and metastatic disease. Half of all PDAC patients have been shown to have loss of SMAD4, which has been shown to correlate with metastasis. Here we functionally demonstrate that SMAD4 is a key suppressor of metastatic colonization of PDAC. Using an in vitro and in vivo multi-component RNA-seq analysis on isogenic human PDAC cell lines, we defined SMAD4-dependent gene expression changes. Specifically, we identified genes that were suppressed in the presence of SMAD4. These genes could contribute to tumor metastasis. To test this, we performed a pooled open reading frame (ORF) overexpression lung colonization assay with the identified genes. We found that expression of the transcription factor FOSL1 is sufficient for metastatic colonization. Mechanistically, SMAD4 directly binds and modulates the activity of the FOSL1 enhancer. Taken together, these studies demonstrate a direct role for SMAD4 in regulating metastatic colonization and identify FOSL1 as a direct SMAD4-regulated gene involved in distant site colonization. Citation Format: Jonathan P. Rennhack, Chao Dai, Andrew Aguirre, John Doench, David Root, William C. Hahn. SMAD4 represses PDAC metastatic colonization through binding FOSL1 enhancer regions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2402.</jats:p