Sree C. Yaddanapudi

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.

BRCA1 and 53BP1 play decisive roles in the choice of DNA double-strand break repair mechanisms. BRCA1 promotes DNA end resection and homologous recombination (HR) during S/G 2 phases of the cell cycle, while 53BP1 inhibits end resection and facilitates non-homologous end-joining (NHEJ), primarily during G 1. This competitive relationship is critical for genome integrity during cell division. However, their relationship in the many cells in our body that are not cycling is unknown. We discovered profound differences in 53BP1 and BRCA1 regulation between cycling and non-cycling cells. Cellular growth arrest results in transcriptional downregulation of BRCA1 and activation of cathepsin-L (CTSL)-mediated degradation of 53BP1. Accordingly, growth-arrested cells do not form BRCA1 or 53BP1 ionizing radiation-induced foci (IRIF). Interestingly, cell cycle re-entry reverts this scenario, with upregulation of BRCA1, downregulation of CTSL, stabilization of 53BP1, and 53BP1 IRIF formation throughout the cycle, indicating that BRCA1 and 53BP1 are important in replicating cells and dispensable in non-cycling cells. We show that CTSL-mediated degradation of 53BP1, previously associated with aggressive breast cancers, is an endogenous mechanism of non-cycling cells to balance NHEJ (53BP1) and HR (BRCA1). Breast cancer cells exploit this mechanism to ensure genome stability and viability, providing an opportunity for targeted therapy.

Ribosomes are vital to the survival of a cell, as they are directly responsible for the synthesis of proteins, which perform critical cellular functions. As such, majority of the energy reserves in a proliferating cell are expended towards synthesis of ribosomes. Cancer cells, with their enhanced proliferation rates, tend to upregulate ribosome biogenesis in order to meet the demand for increased protein synthesis necessary to sustain rapid proliferation. Many of the oncogenes and tumor suppressors known to be deregulated in cancers are capable of positively and negatively regulating ribosome biogenesis, respectively. The ARF tumor suppressor strongly suppresses ribosome biogenesis, particularly in presence of oncogenic signaling. Furthermore, ARF is capable of negatively regulating multiple oncogenes capable of driving tumorigenesis partly through the ribosome biogenesis pathway. As ARF loss is a frequent occurrence in cancer cells, delineating the ARF-regulatory network and determining the impact of ARF loss on this network can give significant insight into the biology of ARF-deficient tumor cells. Expression of the RNase III enzyme, Drosha, has been reported to have prognostic value in multiple cancers. However, Drosha expression appears to have a dual nature in tumorigenesis, as both overexpression and loss of Drosha have been reported to have tumorigenic functions. Although the mechanistic basis of this apparent duality are not yet known, gaining a deeper understanding of Drosha's functional capabilities can give us an insight into its role in tumorigenesis. Drosha performs critical functions in biogenesis of multiple RNA species within the cell, including ribosomal RNA (rRNA), micro RNA (miRNA) and messenger RNA (mRNA). Drosha's role in miRNA biogenesis is the most studied and characterized aspect of its functions and can explain the tumor suppressive aspect of its dual nature; a global decrease in miRNAs has been reported to be part of tumor progression, and loss of Drosha has the potential to significantly deplete mature miRNA population within the cell. However, how overexpression of Drosha can drive tumorigenesis remains to be studied. As enhanced ribosome biogenesis is another feature of cancer cells and Drosha has been shown to aid in processing of r RNA, Drosha's role in ribosome biogenesis pathway has the potential to function in an oncogenic manner. Therefore, further characterization of Drosha's role in ribosome biogenesis can significantly enhance our understanding of its contribution to tumorigenesis. Recent studies in mouse cell lines revealed that ARF tumor suppressor is capable of negatively regulating Drosha expression in a translation-dependent manner. Given the entrenched role of ARF in inhibiting ribosome biogenesis, I hypothesized that ARF's ability to regulate Drosha could impact Drosha's functions in ribosome biogenesis pathway. I further hypothesized that Drosha overexpression could function in a pro-proliferative manner through the ribosome biogenesis pathway. The data presented in this Dissertation reveals that human p14ARF is capable of regulating Drosha protein expression in a dynamic and localized fashion; loss of ARF increases over all cellular Drosha protein levels and also the localization of Drosha to the nucleolus. ARF potentially regulates nucleolar localization of Drosha by sequestering it away from nucleolus, as we found that ARF immunoprecipitated with Drosha in RNA-independent manner. Furthermore, loss of ARF enhances ribosome biogenesis both at the level of 47s rRNA transcription and processing. Association of Drosha with precursor rRNAs was also enhanced in absence of ARF, suggesting that enhanced nucleolar localization of Drosha upon ARF loss contributes to rRNA processing. Drosha overexpression by itself was able to increase ribosome biogenesis, with a modest increase in 47s rRNA transcription and a faster accumulation of 28s and 18s rRNAs. Drosha overexpression led to an increase in ARF expression, although this induction of ARF was not sufficient to inhibit Drosha's ability to enhance ribosome biogenesis and cell proliferation. However, overexpression of ARF negated proliferative enhancement induced by Drosha overexpression. These results point towards a cross-regulatory loop between ARF and Drosha, with functional impact on ribosome biogenesis.