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INTRODUCTION: Oral cavity squamous cell carcinoma (OSCC) occurs most frequently in patients >60 years old with a history of tobacco and alcohol use. Epidemiological studies describe increased incidence of OSCC in younger adults (years). Despite its poor prognosis, knowledge of OSCC tumor microenvironment (TME) characteristics in younger adults is scarce and could help inform possible resistance to emerging treatment options. METHODS: Patients with OSCC were evaluated using TCGA-HNSC (n=121) and a stage and subsite-matched institutional cohort (n=8) to identify differential gene expression focusing on the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) processes in younger (≤45 years) vs. older adults (≥60 years). NanoString nCounter analysis was performed using isolated total RNA from formalin-fixed paraffin-embedded (FFPE) tumor samples. Stained tumor slides from young and old OSCC patients were evaluated for CD8+ T-cell counts using immunohistochemistry. RESULTS: Younger OSCC patients demonstrated significantly increased expression of ECM remodeling and EMT process genes, as well as TME immunosuppression. Gene set enrichment analyses demonstrated increased ECM pathways and concurrent decreased immune pathways in young relative to old patients. Transcripts per million of genetic markers involved in ECM remodeling including LAMB3, VCAN, S100A9, COL5A1, and ITGB2 were significantly increased in tumors of younger vs. older patients (adjusted p-value < 0.10). Young patient TMEs demonstrated a 2.5-fold reduction in CD8+ T-cells as compared to older patients (p < 0.05). CONCLUSION: Differential gene expression impacting ECM remodeling and TME immunosuppression may contribute to disease progression in younger adult OSCC and has implications on response to evolving treatment modalities, such as immune checkpoint inhibitor therapy.

This article describes how the transcriptional alterations of the innate immune system divide dysplasias into aggressive forms that, despite the treatment, relapse quickly and more easily, and others where the progression is slow and more treatable. It elaborates on how the immune system can change the extracellular matrix, favoring neoplastic progression, and how infections can enhance disease progression by increasing epithelial damage due to the loss of surface immunoglobulin and amplifying the inflammatory response. We investigated whether these dysregulated genes were linked to disease progression, delay, or recovery. These transcriptional alterations were observed using the RNA-based next-generation sequencing (NGS) panel Oncomine Immune Response Research Assay (OIRRA) to measure the expression of genes associated with lymphocyte regulation, cytokine signaling, lymphocyte markers, and checkpoint pathways. During the analysis, it became apparent that certain alterations divide dysplasia into two categories: progressive or not. In the future, these biological alterations are the first step to provide new treatment modalities with different classes of drugs currently in use in a systemic or local approach, including classical chemotherapy drugs such as cisplatin and fluorouracile, older drugs like fenretinide, and new checkpoint inhibitor drugs such as nivolumab and pembrolizumab, as well as newer options like T cell therapy (CAR-T). Following these observed alterations, it is possible to differentiate which dysplasias progress or not or relapse quickly. This information could, in the future, be the basis for determining a close follow-up, minimizing surgical interventions, planning a correct and personalized treatment protocol for each patient and, after specific clinical trials, tailoring new drug treatments.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAFV600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation – features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of “minority mitochondrial outer membrane permeabilization (MOMP)” – a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in m

While the emergence of immunotherapies has fundamentally altered the management of solid tumors, cancers exploit many complex biological mechanisms that result in resistance to these agents. These encompass a broad range of cellular activities - from modification of traditional paradigms of immunity via antigen presentation and immunoregulation to metabolic modifications and manipulation of the tumor microenvironment. Intervening on these intricate processes may provide clinical benefit in patients with solid tumors by overcoming resistance to immunotherapies, which is why it has become an area of tremendous research interest with practice-changing implications. This review details the major ways cancers avoid both natural immunity and immunotherapies through primary (innate) and secondary (acquired) mechanisms of resistance, and it considers available and emerging therapeutic approaches to overcoming immunotherapy resistance.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Breast cancer is the most frequently diagnosed malignancy in women worldwide, with tumour metastasis being the leading cause of death for these patients. We have improved our understanding of metastatic disease by studying the role of plasticity in tumour heterogeneity, dissemination, and therapeutic resistance. Yet, metastatic breast cancer remains a burden. The development of successful targeted therapies has been limited by a lack of mechanistic insight into the regulation of cellular adaptability. Next generation sequencing platforms can comprehensively profile the molecules that orchestrate adaptability, but the remaining challenge lies in our ability to computationally reconstruct these plastic mechanisms. It is not yet clear which computational approaches facilitate the reconstruction of plastic mechanisms or what interpretations can be drawn from current data analysis paradigms. Particularly with the emergence of single-cell RNA-sequencing (scRNA-seq) technologies, it is necessary to assess how computational approaches should be coordinated with experimental strategies. In this thesis, I assess the value of both single-cell and bulk sequencing approaches and explore new analytical paradigms that address the continuous nature of cellular plasticity. In doing so, I propose mechanisms of plasticity in bulk and single- cell transcriptomic datasets that may contribute to breast cancer metastasis. First, I investigated mechanisms of metastatic organotropism using bulk RNA-seq data from an in-house optical-barcoding model of breast cancer progression. In this first chapter, I demonstrate the value of traditional bulk approaches in providing clinically-relevant insights, which I then contrast to a single-cell analysis of phenotypic plasticity in the next chapter. Here, I use scRNA-seq data from malignant pleural effusions (MPEs) to disentangle cell-cell interactions between invading breast cancer cells and resident mesothelial populations in this unique microenviron

In chapter one, a general introduction to the basic principles and techniques of MS-based proteomics, quantification strategies, and a generalized shotgun proteomics workflow are given. Moreover, I also outline how to analyze proteomics data from a bioinformatics perspective including normalization, dealing with missing values, differential analysis, functional annotation, as well as how to reveal the biology from post-translational modification data. Furthermore, I generalized the basics of machine learning algorithms from the perspective of supervised and unsupervised machine learning, along with that the application of machine learning algorithms to the identification of protein complexes. In chapter two, we are seeking to explore the drug addiction mechanism in melanoma cells that carry BRAF mutation. We present a proteomics and phosphoproteomics study of BRAFi-addicted melanoma cells (i.e., 451Lu cell line) in response to BRAFi withdrawal, in which ERK1, ERK2, and JUNB were genetically silenced separately using CRISPR-Cas9. We show that inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our data indicate that ERK2 and JUNB share comparable proteome responses dominated by the reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, we reveal that PIR acts as an effector of ERK2, and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to the amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma. In chapter three, we are dedicated to exploring the role of PD-1 in T cell activation by comparing the proteome and phosphoprot

Eukaryotic gene expression is an essential process for proper cell differentiation, development, and the cell’s response to environmental signals. Pre-mRNA splicing is an important part of the eukaryotic gene expression program. Splicing contributes to protein diversity, gene expression regulation, evolution, and genetic diseases. Understanding the intricacies of pre-mRNA splicing is important for understanding gene expression and for the rational design and development of new therapies for genetic diseases.The regulation of pre-mRNA splicing is a highly combinatorial process that relies on many cis- and trans-acting elements. Some of these elements include splice site strength and the intron-exon architecture. It is proposed that spliceosome assembly can either occur across the intron (referred to as intron definition) and across the exon (referred to as exon definition). Selecting between these modes of spliceosome assembly is thought to be dictated by intron architecture. Other studies have demonstrated that the proximity between the 5' splice site and its intronic 3' splice site plays a critical role in splice site selection. In chapter 2 we conducted a genome-wide computational analyses to evaluate the proximity rules in the context of intron and exon definition. Our computational studies were complemented using designer mini-genes in cell transfection assays to evaluate the impact of splice site proximity in alternative splicing. The ability to regulate gene expression allows the cell to adjust to its ever-changing needs and external cues. Aberrant regulation of gene expression is linked to diseases such as cancer. One major contributor to modulate gene expression is through the regulation of mRNA stability. A change in mRNA stability can lead to differing protein expression levels while alternative splicing primarily promotes protein diversity. The work described in Chapter 3 outlines what gene features impact mRNA stability and how alternative splicing can i

Cellular responses to DNA damage constitute one of the most important fields in cancer biology. In this class we will analyze classical and recent papers from the primary research literature to gain a profound understand of cell cycle regulation and DNA damage checkpoints that act as powerful emergency brakes to prevent cancer. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

BRG1 is mutated in 10% of lung cancers, making this mutation clinically relevant. The downstream effects of BRG1 included significantly affecting the cell cycle control and chromosomal replication pathway. CDK18, a cyclin-dependent kinase, was determined to be the gene with significantly decreased expression (p

A small percentage of bladder cancers in the general population have been found to harbor DNA viruses. In contrast, up to 25% of tumors of solid organ transplant recipients, who are at an increased risk of developing bladder cancer and have an overall poorer outcomes, harbor BK polyomavirus (BKPyV). To better understand the biology of the tumors and the mechanisms of carcinogenesis from potential oncoviruses, we performed whole genome and transcriptome sequencing on bladder cancer specimens from 43 transplant patients. Nearly half of the tumors from this patient population contained viral sequences. The most common were from BKPyV (N=9, 21%), JC polyomavirus (N=7, 16%), carcinogenic human papillomaviruses (N=3, 7%), and torque teno viruses (N=5, 12%). Immunohistochemistry revealed variable Large T antigen expression in BKPyV-positive tumors ranging from 100% positive staining of tumor tissue to less than 1%. In most cases of BKPyV-positive tumors, the viral genome appeared to be clonally integrated into the host chromosome consistent with microhomology-mediated end joining and coincided with focal amplifications of the tumor genome similar to other virus-mediated cancers. Significant changes in host gene expression consistent with the functions of BKPyV Large T antigen were also observed in these tumors. Lastly, we identified four mutation signatures in our cases, with those attributable to APOBEC3 and SBS5 being the most abundant. Mutation signatures associated with an antiviral drug, ganciclovir, and aristolochic acid, a nephrotoxic compound found in some herbal medicines, were also observed. The results suggest multiple pathways to carcinogenesis in solid organ transplant recipients with a large fraction being virus-associated.

A high level of homocysteine (Hcy) is associated with oxidative/ER stress, apoptosis, and impairment of angiogenesis, whereas hydrogen sulfide (H2S) has been found to reverse this condition. Recent studies have shown that cancer cells need to produce a high level of endogenous H2S to maintain cell proliferation, growth, viability, and migration. However, any novel mechanism that targets this balance of Hcy and H2S production has yet to be discovered or exploited. Cells require homocysteine metabolism via the methionine cycle for nucleotide synthesis, methylation, and reductive metabolism, and this pathway supports the high proliferative rate of cancer cells. Although the methionine cycle favors cancer cells for their survival and growth, this metabolism produces a massive amount of toxic Hcy that somehow cancer cells handle very well. Recently, research showed specific pathways important for balancing the antioxidative defense through H2S production in cancer cells. This review discusses the relationship between Hcy metabolism and the antiapoptotic, antioxidative, anti-inflammatory, and angiogenic effects of H2S in different cancer types. It also summarizes the historical understanding of targeting antioxidative defense systems, angiogenesis, and other protective mechanisms of cancer cells and the role of H2S production in the genesis, progression, and metastasis of cancer. This review defines a nexus of diet and precision medicine in targeting the delicate antioxidative system of cancer and explores possible future therapeutics that could exploit the Hcy and H2S balance.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Chronic myeloid leukemia (CML) is a blood cancer characterized by dysregulated production of maturing myeloid cells driven by the product of the Philadelphia chromosome, the BCR-ABL1 tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have proved effective in treating CML, but there is still a cohort of patients who do not respond to TKI therapy even in the absence of mutations in the BCR-ABL1 kinase domain that mediate drug resistance. To discover novel strategies to improve TKI therapy in CML, we developed a nonlinear mathematical model of CML hematopoiesis that incorporates feedback control and lineage branching. Cell-cell interactions were constrained using an automated model selection method together with previous observations and new in vivo data from a chimeric BCR-ABL1 transgenic mouse model of CML. The resulting quantitative model captures the dynamics of normal and CML cells at various stages of the disease and exhibits variable responses to TKI treatment, consistent with those of CML patients. The model predicts that an increase in the proportion of CML stem cells in the bone marrow would decrease the tendency of the disease to respond to TKI therapy, in concordance with clinical data and confirmed experimentally in mice. The model further suggests that, under our assumed similarities between normal and leukemic cells, a key predictor of refractory response to TKI treatment is an increased maximum probability of self-renewal of normal hematopoietic stem cells. We use these insights to develop a clinical prognostic criterion to predict the efficacy of TKI treatment and design strategies to improve treatment response. The model predicts that stimulating the differentiation of leukemic stem cells while applying TKI therapy can significantly improve treatment outcomes.

BRG1 is mutated in 10% of lung cancers, making this mutation clinically relevant. The downstream effects of BRG1 included significantly affecting the cell cycle control and chromosomal replication pathway. CDK18, a cyclin-dependent kinase, was determined to be the gene with significantly decreased expression (p

Lipids are one of life’s four main macromolecules and provide essential functions to cells, as they serve as building blocks of cellular membranes, mediate cell signaling, and are a critical energy source. Within cells, esterified fatty acids aggregate into lipid droplets in the form of triglycerides, consisting of a neutral lipid core surrounded by a phospholipid monolayer decorated with various proteins. The degradation of lipid droplets and mobilization of lipids occurs through lipolysis, a process that hydrolyzes a molecule of triglyceride into a glycerol and three fatty acids. Emerging evidence demonstrates the involvement of a selective form of autophagy in lipolysis with two similar—but distinct—mechanisms. The first is lipophagy, in which autophagosomes trap lipid droplets and then fuse with lysosomes. The second is microlipophagy, which occurs when lysosomes directly engulf lipid droplets. Because the homeostatic balance of lipids is critical to proper cellular functions, and whose impairment leads to disease, it is essential to understand how lipid turnover is maintained. The process of microlipophagy has only recently been described, and many features of its regulation are still poorly understood. Our recent study showed that inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway by depletion of Raptor in adipocytes led to an influx of lysosomes and accumulation of lipid droplets within lysosome. Here, we expand on these previous findings and explore whether lysosome induction possibly pertaining to microlipophagy is regulated by mTORC1 or beta adrenergic receptor (BAR), two pathways that are involved in regulating lipid droplet breakdown in adipocytes. We were able to conclude that more lysosomes were induced when BARs are activated rather than by mTORC1 inhibition in primary differentiated adipocytes. Thus, BAR activation rather than mTORC1 may be the primary regulator of microlipophagy. This research could help elucidate the dyna

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

Cellular responses to DNA damage constitute one of the most important fields in cancer biology. In this class we will analyze classical and recent papers from the primary research literature to gain a profound understand of cell cycle regulation and DNA damage checkpoints that act as powerful emergency brakes to prevent cancer. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. In 1971, President Nixon declared the "War on Cancer," but after three decades the war is still raging. How much progress have we made toward winning the war and what are we doing to improve the fight? Understanding the molecular and cellular events involved in tumor formation, progression, and metastasis is crucial to the development of innovative therapy for cancer patients. Insights into these processes have been gleaned through basic research using biochemical, molecular, and genetic analysis in yeast, C. elegans, mice, and cell culture models. We will explore the laboratory tools and techniques used to perform cancer research, major discoveries in cancer biology, and the medical implications of these breakthroughs. A focus of the class will be critical analysis of the primary literature to foster understanding of the strengths and limitations of various approaches to cancer research. Special attention will be made to the clinical implications of cancer research performed in model organisms and the prospects for ending the battle with this devastating disease.

This course will act as an introduction to the latest developments in the fields of cancer biology and immunotherapy. Almost everyone knows someone whose life has been affected by cancer. Why is cancer such a difficult disease to treat? What is the best system to model the development of a human tumor? How can new treatment modalities, especially immune-based therapies that harness the natural ability of immune cells to kill target cells, be developed to treat cancer? These and other questions will be addressed in this course. We will explore the concepts of mouse models for human cancer, humanized cancer mice and cancer immunotherapy by reading recent and classic research articles. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. In 1971, President Nixon declared the "War on Cancer," but after three decades the war is still raging. How much progress have we made toward winning the war and what are we doing to improve the fight? Understanding the molecular and cellular events involved in tumor formation, progression, and metastasis is crucial to the development of innovative therapy for cancer patients. Insights into these processes have been gleaned through basic research using biochemical, molecular, and genetic analysis in yeast, C. elegans, mice, and cell culture models. We will explore the laboratory tools and techniques used to perform cancer research, major discoveries in cancer biology, and the medical implications of these breakthroughs. A focus of the class will be critical analysis of the primary literature to foster understanding of the strengths and limitations of various approaches to cancer research. Special attention will be made to the clinical implications of cancer research performed in model organisms and the prospects for ending the battle with this devastating disease.

This course will act as an introduction to the latest developments in the fields of cancer biology and immunotherapy. Almost everyone knows someone whose life has been affected by cancer. Why is cancer such a difficult disease to treat? What is the best system to model the development of a human tumor? How can new treatment modalities, especially immune-based therapies that harness the natural ability of immune cells to kill target cells, be developed to treat cancer? These and other questions will be addressed in this course. We will explore the concepts of mouse models for human cancer, humanized cancer mice and cancer immunotherapy by reading recent and classic research articles. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

The research focus of this Doctoral Thesis is to study the following unanswered questions related to NCOA4 and its central role in iron biology under healthy and disease conditions. In Chapter 1 we review the biology of NCOA4, its physiological role and its potential role in neurodegenerative disorders (ND). Many NDs have been associated with inappropriate iron accumulation in areas most susceptible to disease leading to oxidative stress, altered ferritin levels, and abnormal decreases in the autophagy-lysosomal pathway. Chapter 2 investigates the functional role of NCOA4-mediated ferritinophagy in regulating systemic iron homeostasis and the erythroid cell autonomous role of NCOA4 in erythropoiesis. Chapter 3 evaluates the non-autonomous contribution of NCOA4 to erythropoiesis by NCOA4 function in macrophages. Next, in Chapter 4 we studied long-term liver iron overload using mouse models of iron overload to understand the cellular pathways that lead to pathological changes or those that may provide protection against damage. To achieve this, we employ a state-of the art mass spectrometry-based quantitative proteomics approach. Chapter 5 is based on an exciting finding from chapter 4 where we identify a new role for NCOA4-mediated ferritinophagy during long-term iron overload in vivo. In Chapter 6 a new role for ferritinophagy is discovered in pancreatic cancer pathogenesis. Last, the primary tool utilized in the experimental methods supporting this Doctoral Thesis is proteomics, a high-throughput technology. As highlighted in Chapter 7, multi-omics approaches have helped to address important questions regarding the etiology and genetic/molecular background of different cancers at a level not otherwise attainable with conventional methods. However, we reflect on the need to recognize the potential harms of face value interpretation of these high-throughput analyses. Ethical concerns may arise when some of these large-scale omics studies fail to represent the broader

Pancreatic cancer is one of the deadliest cancer types with poor treatment options. Better detection of early symptoms and relevant disease correlations could improve pancreatic cancer prognosis. In this retrospective study, we used symptom and disease codes (ICD-10) from the Danish National Patient Registry (NPR) encompassing 6.9 million patients from 1994 to 2018,, of whom 23,592 were diagnosed with pancreatic cancer. The Danish cancer registry included 18,523 of these patients. To complement and compare the registry diagnosis codes with deeper clinical data, we used a text mining approach to extract symptoms from free text clinical notes in electronic health records (3078 pancreatic cancer patients and 30,780 controls). We used both data sources to generate and compare symptom disease trajectories to uncover temporal patterns of symptoms prior to pancreatic cancer diagnosis for the same patients. We show that the text mining of the clinical notes was able to complement the registry-based symptoms by capturing more symptoms prior to pancreatic cancer diagnosis. For example, 'Blood pressure reading without diagnosis', 'Abnormalities of heartbeat', and 'Intestinal obstruction' were not found for the registry-based analysis. Chaining symptoms together in trajectories identified two groups of patients with lower median survival (<90 days) following the trajectories 'Cough→Jaundice→Intestinal obstruction' and 'Pain→Jaundice→Abnormal results of function studies'. These results provide a comprehensive comparison of the two types of pancreatic cancer symptom trajectories, which in combination can leverage the full potential of the health data and ultimately provide a fuller picture for detection of early risk factors for pancreatic cancer.

Pancreatic cancer is one of the deadliest cancer types with poor treatment options. Better detection of early symptoms and relevant disease correlations could improve pancreatic cancer prognosis. In this retrospective study, we used symptom and disease codes (ICD-10) from the Danish National Patient Registry (NPR) encompassing 6.9 million patients from 1994 to 2018,, of whom 23,592 were diagnosed with pancreatic cancer. The Danish cancer registry included 18,523 of these patients. To complement and compare the registry diagnosis codes with deeper clinical data, we used a text mining approach to extract symptoms from free text clinical notes in electronic health records (3078 pancreatic cancer patients and 30,780 controls). We used both data sources to generate and compare symptom disease trajectories to uncover temporal patterns of symptoms prior to pancreatic cancer diagnosis for the same patients. We show that the text mining of the clinical notes was able to complement the registry-based symptoms by capturing more symptoms prior to pancreatic cancer diagnosis. For example, 'Blood pressure reading without diagnosis', 'Abnormalities of heartbeat', and 'Intestinal obstruction' were not found for the registry-based analysis. Chaining symptoms together in trajectories identified two groups of patients with lower median survival (<90 days) following the trajectories 'Cough→Jaundice→Intestinal obstruction' and 'Pain→Jaundice→Abnormal results of function studies'. These results provide a comprehensive comparison of the two types of pancreatic cancer symptom trajectories, which in combination can leverage the full potential of the health data and ultimately provide a fuller picture for detection of early risk factors for pancreatic cancer.

Background: Antibodies against the programmed death-1 (PD-1) receptor and its ligand (PD-L1) have been recently approved for small-cell lung cancer (SCLC) treatment. Circulating tumour cells (CTCs) have emerged as an appealing liquid biopsy candidate that could enhance treatment decision-making in systemic therapy for SCLC patients. Several current technologies enrich CTCs using specific surface epitopes, size, rigidity, or dielectric properties. However, they are hampered by the heterogeneity of the enriched cells from blood samples. Methods: We evaluated two CTC enrichment systems: EpCAM conjugated to magnetic beads and a microfluidic device (Parsortix, Angle plc). PD-L1 expression was evaluated on the isolated CTCs. Twenty-three blood samples were collected from 21 patients with SCLC. PD-L1 expression was determined on CTCs through immunofluorescent staining. Results: CTCs were found in 14/23 (60.9%) of the samples, with 11/23 (47.8%) through EpCAM-coated magnetic beads (range, 4–1,611 CTCs/8 mL; median =5) and 11/20 (55.0%) using the Parsortix system (range, 1–165 CTCs/8 mL; median =4). Notably, a total of 17 EpCAM-negative CTCs were isolated using the Parsortix system. PD-L1 expression was detected on 268 of the 3,501 (7.7%) CTCs isolated with EpCAM-coated beads and in 33/366 (9.0%) of the CTCs isolated with the Parsortix system. No vimentin expression was observed in any of the detected CTCs. Conclusions: Overall, we identified a population of EpCAM-negative SCLC CTCs and showed that PD-L1 expression can be assessed on CTCs from SCLC patients. Comparison to tumour and treatment outcomes is needed to validate the potential of CTCs as an alternative sample for the assessment of PD-L1 expression in SCLC.

In multicellular organisms, survival of the organism is favored over survival of individual cells. As such, normal cells are subject to limits on proliferation. In cancer, however, the accumulation of somatic, and sometimes germline, alterations converges to produce cells which are not subject to or can bypass normal growth restrictions, thus enabling the general cancer phenotype of dysregulated and excessive cell growth. Over the decades, we have made significant gains towards understanding how individual genetic alterations affect cell biology, and how these effects can converge to produce the cancer phenotype. Due to the cancer’s immense heterogeneity, however, we have yet to fully understand the whole-cell dynamic properties leading to the diversity of cancer sub-phenotypes that are observed in patients.The major theme of this dissertation is to interrogate many genetic backgrounds to characterize biological processes that are critical in the formation and/or maintenance of the general cancer phenotype. In chapter one, I performed a genome-wide screen to characterize the ultraviolet light-induced DNA damage response in the model organism Saccharomyces cerevisiae using a metric which reflects a time-dependent growth phenotype. This metric allowed me to identify a set of mitochondrial-associated genes involved in the response to ultraviolet-induced DNA damage. In chapter two, I used a context-aware deep learning model of therapeutic response to examine the cellular response to palbociclib, a selective inhibitor of the cyclin-dependent kinases four and six. I found that the response to palbociclib is governed by an array of distinct biological processes, and that patients and cell line samples are best stratified with the integration of all of these pathways. Overall, this body of work uses specific measures of context to further characterize biological processes critical to the development and maintenance of the cancer phenotype.

With the advent of immunotherapy, identification and characterization of cancer-specific antigens is paramount to our ability to leverage this field towards the treatment of cancer. Pursuing the study of these cell surface antigens, especially in the context of cellular stress has become increasingly important. Low oxygen availability, or hypoxia, is a major environmental factor in solid tumors, which influences the metabolic, transcriptomic, and proteomic landscape of these cells. Hypoxia ultimately leads to poorer prognoses for patients with hypoxic solid tumors, and these hypoxia-specific effects suggest that tumor hypoxia can be leveraged for early stage diagnostics, as well as selective targeting of hypoxia-induced antigens in tumors. There are two main modes by which cell surface targets can be leveraged for the selective targeting of diseased cells, namely (1) through direct binding of a tumor cell surface antigen by antibodies and downstream activation of surrounding immune cells or (2) through the interaction of MHC-I peptide complexes with the native T-cell receptor (TCR). This work encompasses novel methods for the identification of both whole protein and MHC-peptide complexes in the context of hypoxic pancreatic cancer. As pancreatic cancer continues to lack tangible treatment options, it has become increasingly important to characterize these tumor types for new and selective therapeutic targets. In Chapter 1, we perform cell surface proteomics on an array of basal and classical pancreatic adenocarcinoma (PDAC) cells in hypoxia and normal conditions and identify the membrane protein vasorin (VASN) as a potential marker for hypoxic PDAC. In Chapter 2, we develop a novel method for small-scale cell surface proteomics using membrane tethered forms of the promiscuous biotinylators APEX2 and HRP. In Chapter 3, we design a novel secreted HLA Fc-fusion construct that can be coupled with mass spectrometry to profile the immunopeptidome of disease-phenotypes, in

Aspirin intake has been shown to lead to significant protection against colorectal cancer, for example with an up to twofold reduction in colorectal adenoma incidence rates at higher doses. The mechanisms contributing to protection are not yet fully understood. While aspirin is an anti-inflammatory drug and can thus influence the tumor microenvironment, in vitro and in vivo experiments have recently shown that aspirin can also have a direct effect on cellular kinetics and fitness. It reduces the rate of tumor cell division and increases the rate of cell death. The question arises whether such changes in cellular fitness are sufficient to significantly contribute to the epidemiologically observed protection. To investigate this, we constructed a class of mathematical models of in vivo evolution of advanced adenomas, parameterized it with available estimates, and calculated population level incidence. Fitting the predictions to age incidence data revealed that only a model that included colonic crypt competition can account for the observed age-incidence curve. This model was then used to predict modified incidence patterns if cellular kinetics were altered as a result of aspirin treatment. We found that changes in cellular fitness that were within the experimentally observed ranges could reduce advanced adenoma incidence by a sufficient amount to account for age incidence data in aspirin-treated patient cohorts. While the mechanisms that contribute to the protective effect of aspirin are likely complex and multi-factorial, our study demonstrates that direct aspirin-induced changes of tumor cell fitness can significantly contribute to epidemiologically observed reduced incidence patterns.

Background: Antibodies against the programmed death-1 (PD-1) receptor and its ligand (PD-L1) have been recently approved for small-cell lung cancer (SCLC) treatment. Circulating tumour cells (CTCs) have emerged as an appealing liquid biopsy candidate that could enhance treatment decision-making in systemic therapy for SCLC patients. Several current technologies enrich CTCs using specific surface epitopes, size, rigidity, or dielectric properties. However, they are hampered by the heterogeneity of the enriched cells from blood samples. Methods: We evaluated two CTC enrichment systems: EpCAM conjugated to magnetic beads and a microfluidic device (Parsortix, Angle plc). PD-L1 expression was evaluated on the isolated CTCs. Twenty-three blood samples were collected from 21 patients with SCLC. PD-L1 expression was determined on CTCs through immunofluorescent staining. Results: CTCs were found in 14/23 (60.9%) of the samples, with 11/23 (47.8%) through EpCAM-coated magnetic beads (range, 4–1,611 CTCs/8 mL; median =5) and 11/20 (55.0%) using the Parsortix system (range, 1–165 CTCs/8 mL; median =4). Notably, a total of 17 EpCAM-negative CTCs were isolated using the Parsortix system. PD-L1 expression was detected on 268 of the 3,501 (7.7%) CTCs isolated with EpCAM-coated beads and in 33/366 (9.0%) of the CTCs isolated with the Parsortix system. No vimentin expression was observed in any of the detected CTCs. Conclusions: Overall, we identified a population of EpCAM-negative SCLC CTCs and showed that PD-L1 expression can be assessed on CTCs from SCLC patients. Comparison to tumour and treatment outcomes is needed to validate the potential of CTCs as an alternative sample for the assessment of PD-L1 expression in SCLC.

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and t

Deep learning methods have significantly advanced the state of computer vision and natural language processing. Their ability to discover intricate patterns in ever-expanding datasets is critical in solving cancer biology problems. However, cancer biology poses unique challenges. Typical input data, such as tumor images and DNA sequences, have significantly different semantic contexts than the traditional datasets used to train the deep learning methods. Thus, it is infeasible to leverage large pre-trained networks and requires training from scratch. Moreover, these data types are not human readable, making it difficult to annotate the data and interpret what the model has learned. This thesis aims to resolve these challenges and solve three urgent cancer biology problems using deep learning methods.Cancer is mediated through various mechanisms. One such mechanism is circular extrachromosomal DNA (ecDNA), one of the primary drivers of oncogene amplification. EcDNA is prevalent across a wide variety of cancer types and leads to worse patient survival. Thus, there is a critical need for tools to study these genomic lesions. However, it is difficult to understand various facets of ecDNA just through sequence-based methods and requires image-based reconstructions. I first present ecSeg, a deep learning tool to reconstruct ecDNA in images of tumor cells in metaphase. EcSeg uses a fully convolutional network and traditional computer vision techniques to semantically segment ecDNA. EcSeg correlates these segmentations with amplification profiles to reveal ecDNA mechanics and their resistance to drug therapy. To translate ecSeg to clinical practice, I present ecSeg-i to resolve the ecDNA status of interphase cells in cancer patient tissue. Tissue images primarily contain interphase cells in which the DNA is loosely wound, making it extremely challenging to distinguish ecDNA. EcSeg-i uses a DenseNet to determine the ecDNA status and amplification profiles of cancer patient t

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

With the advent of immunotherapy, identification and characterization of cancer-specific antigens is paramount to our ability to leverage this field towards the treatment of cancer. Pursuing the study of these cell surface antigens, especially in the context of cellular stress has become increasingly important. Low oxygen availability, or hypoxia, is a major environmental factor in solid tumors, which influences the metabolic, transcriptomic, and proteomic landscape of these cells. Hypoxia ultimately leads to poorer prognoses for patients with hypoxic solid tumors, and these hypoxia-specific effects suggest that tumor hypoxia can be leveraged for early stage diagnostics, as well as selective targeting of hypoxia-induced antigens in tumors. There are two main modes by which cell surface targets can be leveraged for the selective targeting of diseased cells, namely (1) through direct binding of a tumor cell surface antigen by antibodies and downstream activation of surrounding immune cells or (2) through the interaction of MHC-I peptide complexes with the native T-cell receptor (TCR). This work encompasses novel methods for the identification of both whole protein and MHC-peptide complexes in the context of hypoxic pancreatic cancer. As pancreatic cancer continues to lack tangible treatment options, it has become increasingly important to characterize these tumor types for new and selective therapeutic targets. In Chapter 1, we perform cell surface proteomics on an array of basal and classical pancreatic adenocarcinoma (PDAC) cells in hypoxia and normal conditions and identify the membrane protein vasorin (VASN) as a potential marker for hypoxic PDAC. In Chapter 2, we develop a novel method for small-scale cell surface proteomics using membrane tethered forms of the promiscuous biotinylators APEX2 and HRP. In Chapter 3, we design a novel secreted HLA Fc-fusion construct that can be coupled with mass spectrometry to profile the immunopeptidome of disease-phenotypes, in

In multicellular organisms, survival of the organism is favored over survival of individual cells. As such, normal cells are subject to limits on proliferation. In cancer, however, the accumulation of somatic, and sometimes germline, alterations converges to produce cells which are not subject to or can bypass normal growth restrictions, thus enabling the general cancer phenotype of dysregulated and excessive cell growth. Over the decades, we have made significant gains towards understanding how individual genetic alterations affect cell biology, and how these effects can converge to produce the cancer phenotype. Due to the cancer’s immense heterogeneity, however, we have yet to fully understand the whole-cell dynamic properties leading to the diversity of cancer sub-phenotypes that are observed in patients.The major theme of this dissertation is to interrogate many genetic backgrounds to characterize biological processes that are critical in the formation and/or maintenance of the general cancer phenotype. In chapter one, I performed a genome-wide screen to characterize the ultraviolet light-induced DNA damage response in the model organism Saccharomyces cerevisiae using a metric which reflects a time-dependent growth phenotype. This metric allowed me to identify a set of mitochondrial-associated genes involved in the response to ultraviolet-induced DNA damage. In chapter two, I used a context-aware deep learning model of therapeutic response to examine the cellular response to palbociclib, a selective inhibitor of the cyclin-dependent kinases four and six. I found that the response to palbociclib is governed by an array of distinct biological processes, and that patients and cell line samples are best stratified with the integration of all of these pathways. Overall, this body of work uses specific measures of context to further characterize biological processes critical to the development and maintenance of the cancer phenotype.

Deep learning methods have significantly advanced the state of computer vision and natural language processing. Their ability to discover intricate patterns in ever-expanding datasets is critical in solving cancer biology problems. However, cancer biology poses unique challenges. Typical input data, such as tumor images and DNA sequences, have significantly different semantic contexts than the traditional datasets used to train the deep learning methods. Thus, it is infeasible to leverage large pre-trained networks and requires training from scratch. Moreover, these data types are not human readable, making it difficult to annotate the data and interpret what the model has learned. This thesis aims to resolve these challenges and solve three urgent cancer biology problems using deep learning methods.Cancer is mediated through various mechanisms. One such mechanism is circular extrachromosomal DNA (ecDNA), one of the primary drivers of oncogene amplification. EcDNA is prevalent across a wide variety of cancer types and leads to worse patient survival. Thus, there is a critical need for tools to study these genomic lesions. However, it is difficult to understand various facets of ecDNA just through sequence-based methods and requires image-based reconstructions. I first present ecSeg, a deep learning tool to reconstruct ecDNA in images of tumor cells in metaphase. EcSeg uses a fully convolutional network and traditional computer vision techniques to semantically segment ecDNA. EcSeg correlates these segmentations with amplification profiles to reveal ecDNA mechanics and their resistance to drug therapy. To translate ecSeg to clinical practice, I present ecSeg-i to resolve the ecDNA status of interphase cells in cancer patient tissue. Tissue images primarily contain interphase cells in which the DNA is loosely wound, making it extremely challenging to distinguish ecDNA. EcSeg-i uses a DenseNet to determine the ecDNA status and amplification profiles of cancer patient t

A cancer gene therapy involves the expression of a foreign gene in cancer cells. However, the current methods to control the transgene expression are imperfect, with leaky expression usually found in normal cells. This leakiness can lead to harmful side effects in patients, especially if the transgene encodes a toxic protein. Herein, my studies aim to invent, as well as improve upon regulatory tools to control transgene expression in cancer gene therapies. Compared to the traditional promoter-based or cell surface marker-based approaches, my approach targets an intracellular protein defect caused by several mutations that are prevalent in cancers. Additionally, I improved the inducible gene expression system, further eliminating its background leakiness. Together, my studies offer novel cell-specific targeting and temporal control options, expanding the current gene therapy toolbox.

Aspirin intake has been shown to lead to significant protection against colorectal cancer, for example with an up to twofold reduction in colorectal adenoma incidence rates at higher doses. The mechanisms contributing to protection are not yet fully understood. While aspirin is an anti-inflammatory drug and can thus influence the tumor microenvironment, in vitro and in vivo experiments have recently shown that aspirin can also have a direct effect on cellular kinetics and fitness. It reduces the rate of tumor cell division and increases the rate of cell death. The question arises whether such changes in cellular fitness are sufficient to significantly contribute to the epidemiologically observed protection. To investigate this, we constructed a class of mathematical models of in vivo evolution of advanced adenomas, parameterized it with available estimates, and calculated population level incidence. Fitting the predictions to age incidence data revealed that only a model that included colonic crypt competition can account for the observed age-incidence curve. This model was then used to predict modified incidence patterns if cellular kinetics were altered as a result of aspirin treatment. We found that changes in cellular fitness that were within the experimentally observed ranges could reduce advanced adenoma incidence by a sufficient amount to account for age incidence data in aspirin-treated patient cohorts. While the mechanisms that contribute to the protective effect of aspirin are likely complex and multi-factorial, our study demonstrates that direct aspirin-induced changes of tumor cell fitness can significantly contribute to epidemiologically observed reduced incidence patterns.

Human prostate cancer can result from chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion-independent ETS factor upregulation and prostate oncogenesis remain relatively unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occur through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF-deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion-independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies., source:https://www.frontiersin.org/articles/10.3389/fendo.2022.1042787/full

The pancreatic ductal adenocarcinoma microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAMs) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment. However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEMs) in vitro and performed detailed, multiomic characterization (i.e., transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single-cell RNA sequencing dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K-AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization.

The establishment of polarity within tissues and dynamic cellular morphogenetic events are features common to both developing and adult tissues, and breakdown of these programs is associated with diverse human diseases. Wnt/Planar cell polarity (Wnt/PCP) signaling, a branch of non-canonical Wnt signaling, is critical to the establishment and maintenance of polarity in epithelial tissues as well as cell motility events critical to proper embryonic development. In epithelial tissues, Wnt/PCP-mediated planar polarity relies upon the asymmetric distribution of core proteins to establish polarity, but the requirement for this distribution in Wnt/PCP-mediated cell motility remains unclear. However, in both polarized tissues and migratory cells, the Wnt/PCP-specific transmembrane protein Vangl is required and appears to serve as a scaffold upon which the core pathway components as well as positive and negative regulators of Wnt/PCP signaling assemble. The current literature suggests that the multiple interaction domains of Vangl allow for the binding of diverse signaling partners for the establishment of context- and tissue-specific complexes. In this review we discuss the role of Vangl as a master scaffold for Wnt/PCP signaling in epithelial tissue polarity and cellular motility events in developing and adult tissues, and address how these programs are dysregulated in human disease.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Iron is essential part of the human metabolism. It is a catalytic co-factor for many proteins, but it generates harmful reactive oxygen species in human body. Also, many irons metabolic genes are changes in human cancer cells compared to normal cells, leading to iron accumulation in cancer cells especially in colorectal cancer. Due to iron’s significant role in colorectal cancer promotion, much research is focused on its role in colorectal cancer genesis. But some important metabolic aspects are not fully addressed and researched. One key aspect of iron on colorectal cancer cell progression is hemin iron. Much research warned hemin iron role in colorectal cancer cell promotion, but nobody addressed sestirn2 and hemin iron relationship on tumor progression. Sestrin2 is already known to be highly expressed colorectal tumor and neutralize excess reactive oxygen species in colorectal cancer cells. We found sestrin2 is overexpressed in colorectal cancer cells and promotes colorectal cancer development under hemin iron conditions through generating adequate reactive oxygen species. Next, we are looking for another protein TFRC for iron uptake. TFRC is membrane receptor protein which helps iron influx in the cell. TFRC is highly upregulated in colorectal cancer cells, but its exact mechanism on tumor progression is not fully addressed. We found TFRC disruption or iron restriction deactivates the TNKS activity, which drives beta-catenin signaling and colon tumorigenesis. Importantly, we discovered that the combination of iron chelators and DNA damage agents has a synergistic effect in killing human colorectal cancer cells, which provides a new therapeutic approach. To address TFRC high expression in human colorectal cancer cells, we are looking for pathways, which affects TFRC high expression in human. TFRC is affected by Iron-responsive element-binding protein 2 (IRP2) which expression is also maintained by cellular iron status such as ferritin heavy chains. To d

Arsenite exposure leads to the retention of UV-induced DNA damage, thus burdening translesion synthesis (TLS). Rad18 is an essential factor in initiating TLS through PCNA monoubiquitination and is implicated in homologous recombination. It contains two functionally and structurally distinct zinc fingers that are potential targets for arsenite binding. Results from this study reveal arsenite binding to both zinc fingers of Rad18 and a corresponding loss of domain function. Importantly, arsenite inhibited Rad18 RING-dependent PCNA monoubiquitination and polymerase eta recruitment to DNA damage. Further analysis demonstrated multiple effects of arsenite, including the reduction in the nuclear localization and UV-induced chromatin recruitment of Rad18. Arsenite and Rad18 knockdown in UV exposed keratinocytes significantly increased markers of replication stress and DNA strand breaks to a similar degree, suggesting arsenite mediates its effects through Rad18. Altogether, this dissertation supports a mechanism by which arsenite inhibits TLS through the altered activity and regulation of Rad18.

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies., source:https://www.frontiersin.org/articles/10.3389/fendo.2022.1042787/full