Nirupa Murugaesu

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.

An in vivo functional screen identifies ST6GalNAc2 sialyltransferase as a breast cancer metastasis suppressor.

To interrogate the complex mechanisms involved in the later stages of cancer metastasis, we designed a functional in vivo RNA interference (RNAi) screen combined with next-generation sequencing. Using this approach, we identified the sialyltransferase ST6GalNAc2 as a novel breast cancer metastasis suppressor. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on the tumor cell surface, facilitating binding of the soluble lectin galectin-3. This then enhances tumor cell retention and emboli formation at metastatic sites leading to increased metastatic burden, events that can be completely blocked by galectin-3 inhibition. Critically, elevated ST6GALNAC2, but not galectin-3, expression in estrogen receptor-negative breast cancers significantly correlates with reduced frequency of metastatic events and improved survival. These data demonstrate that the prometastatic role of galectin-3 is regulated by its ability to bind to the tumor cell surface and highlight the potential of monitoring ST6GalNAc2 expression to stratify patients with breast cancer for treatment with galectin-3 inhibitors.

Whole genome in vivo RNAi screening identifies the leukemia inhibitory factor receptor as a novel breast tumor suppressor

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

An in vivo functional screen identifies JNK signaling as a modulator of chemotherapeutic response in breast cancer

Chemotherapy remains the mainstay of treatment for advanced breast cancer; however, resistance is an inevitable event for the majority of patients with metastatic disease. Moreover, there is little information available to guide stratification of first-line chemotherapy, crucial given the common development of multidrug resistance. Here, we describe an in vivo screen to interrogate the response to anthracycline-based chemotherapy in a syngeneic metastatic breast cancer model and identify JNK signaling as a key modulator of chemotherapy response. Combining in vitro and in vivo functional analyses, we demonstrate that JNK inhibition both promotes tumor cell cytostasis and blocks activation of the proapoptotic protein Bax, thereby antagonizing chemotherapy-mediated cytotoxicity. To investigate the clinical relevance of this dual role of JNK signaling, we developed a proliferation-independent JNK activity signature and demonstrate high JNK activity to be enriched in triple-negative and basal-like breast cancer subtypes. Consistent with the dual role of JNK signaling in vitro, high-level JNK pathway activation in triple-negative breast cancers is associated both with poor patient outcome in the absence of chemotherapy treatment and, in neoadjuvant clinical studies, is predictive of enhanced chemotherapy response. These data highlight the potential of monitoring JNK activity as early biomarker of response to chemotherapy and emphasize the importance of rational treatment regimes, particularly when combining cytostatic and chemotherapeutic agents. Mol Cancer Ther; 16(9); 1967–78. ©2017 AACR.

Abstract 3417: An in vivo functional screen to identify metastasis suppressor genes

Background When breast carcinomas remain confined to breast tissue, cure rates exceed 90% (http://seer.cancer.gov/csr/1975_2006/). However, if the cancer disseminates through the body, long-term survival decreases depending upon the extent of, and the sites of, colonisation Genes that control the different stages of the metastatic process need to be identified to better delineate the process, and to aid in the development of metastatic biomarkers and provide potential targets for the treatment of metastatic disease. Genetic screens, such as those that exploit RNA interference (RNAi), provide an unbiased approach to the identification of genes associated with a phenotype of interest. Although cell-based screens have been highly informative in identifying genes involved in tumour cell survival, migration and invasion, these in vitro approaches are largely unsuitable for interrogating the later stages of the metastatic process, in particular the processes of cell dissemination, tumour cell extravasation from the circulation and colonisation of secondary sites. Method Using the 4T1 mouse model an in vivo functional metastasis screen that integrates RNAi technology and massively parallel sequencing was implemented to rapidly discover and validate metastasis genes. Results Using this approach, 12 ‘hits’ that suppress tumour cell colonisation of the lungs were identified. 3 of the top 5 hits have been validated as novel metastasis suppressor genes in both mouse and human cell lines and further studies have been undertaken to elucidate the mechanism of action of the top hit. Critically, these findings are clinically relevant in primary breast cancers where there is a significant correlation between elevated expression levels of these suppressor genes and reduced frequency of metastatic events. Conclusion This study demonstrates the value of adopting an unbiased methodology to discover novel metastatic genes and establishes, for the first time, that an in vivo metastasis screen can be combined with next generation sequencing to identify novel components of the metastatic process. 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 3417. doi:1538-7445.AM2012-3417

Abstract P5-05-02: Whole Genome In Vivo RNA Interference Screening Identifies the Leukemia Inhibitory Factor Receptor as a Novel Breast Tumor Suppressor

Background: Cancer is caused by mutations in oncogenes and tumor suppressor (TS) genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important TS genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced TS genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate TS genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate TS genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation and mammary fat pad xenograft tumor formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated TS genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (p 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 4979. doi:10.1158/1538-7445.AM2011-4979