Cancer Biology

Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer with standard therapy involving neoadjuvant chemotherapy, surgical management, and radiation therapy. However, the high recurrence rate and low pathological complete response of TNBC suggest that radioresistance is a critical factor in the diminished therapeutic efficiency of the current treatment strategy. There is limited literature exploring the specific pathways responsible for radiation resistance in TNBC, but most data support the role of limiting reactive oxygen species (ROS) accumulation. Our lab has studied the role of Vascular Endothelial Growth Factor (VEGF) binding to Neuropilin-2 (NRP2) and initiating several cancer stem cell properties. Preliminary data indicate that radiation enriches for NRP2 expressing cells and using a function-blocking antibody specific to VEGF/NRP2 with irradiation decreases cell viability compared to either treatment alone in a TNBC organoid. The central hypothesis of this proposal is that VEGF/NRP2 induces radioresistance by altering redox homeostasis and can be targeted for better therapeutic outcomes in TNBC. This proposal will seek to investigate the possible role of NRP2 in regulating NOS2 transcription and its contribution to mitigating ROS accumulation. I will also use single-cell RNA sequencing technology to identify the subpopulations of TNBC that are radioresistant and whether they utilize the NRP2/NOS2 signaling axis. Another aspect of this proposal is to observe the effectiveness of a function-blocking antibody of NRP2 with radiation using an in vivo model. I plan to identify if this approach reduces the radioresistant clones in TNBC. The completion of this proposal will heighten the understanding of radioresistance in TNBC and identify a novel molecular pathway responsible for this phenotype.

Mitochondrial function declines during aging. The dysfunction is accelerated in age-associated diseases such as Alzheimer’s Disease and Parkinson’s Disease. Thus, therapeutic approaches to maintain or recover mitochondrial function may promote healthy aging or slow age-associated disease progression. Recently, we have shown that the mitochondrial network expansion that occurs during development is an emergent property of the synthesis of highly expressed mitochondrial proteins. Increased mitochondrial import of the highly expressed mitochondrial proteins outcompete the transcription factor ATFS-1, preventing it from entering mitochondria. This allows ATFS-1 to traffic to the nucleus and activate a mitochondrial network expansion transcription program known as UPRmt. These findings suggest an interplay between protein synthesis, mitochondria protein import capacity, and mitochondrial network expansion. The Integrated Stress Response (ISR) is a translation control pathway that reduces overall protein synthesis while preferentially increases translation of ATF-4 in response to diverse stressors including amino acid depletion, ER dysfunction and mitochondrial perturbations. The ISR is mediated by 4 kinases (3 in C. elegans) that all phosphorylate the translation initiation factor eIF2α, which in turn modulates protein synthesis. While considerable work has demonstrated that the ISR is active in response to mitochondrial perturbation, the functional outputs of the ISR related to mitochondrial biology remain unknown. I have obtained or generated several C. elegans strains in which the ISR is impaired. Quite surprisingly, these worms have increased mitochondrial mass and mitochondrial genomes. Intriguingly, these animals also live significantly longer than wildtype worms, suggesting that increased mitochondrial mass is sufficient to extend organismal lifespan. I hypothesize that the ISR matches mitochondrial network expansion with the physiological and environmental inputs that activate the ISR by antagonizing ATFS-1 function. Here, I focus on the role of ISR-dependent translation attenuation or ATF-4 synthesis as direct or indirect regulators of ATFS-1-dependent transcription via the following aims. 1. Determine the mechanisms by which the ISR regulates ATFS-1-dependent mitochondrial biogenesis. 2. Elucidate the mechanisms by which the ISR modulates longevity and healthspan.

Defects in autophagy, the self-degradation of cellular components, are linked to multiple disorders such as cancer, diabetes and neurodegenerative diseases. Autophagosomes, containing cargo marked for degradation, fuse with lysosomes to recycle cell resources, such as protein aggregates and damaged organelles. However, we know little about the mechanisms that regulate the association between autophagic cargoes and autophagosome formation. Here, I investigate the role of vps13d, an essential gene with relatively unknown function, in context-specific autophagy and cell death in the developing Drosophila intestine. Proteins that regulate autophagy and cell death are of particular interest given the roles they play in tumorigenesis. Previous studies of VPS13D identified a role in the clearance of mitochondria by autophagy, also known as mitophagy. Intriguingly, VPS13D also appears to be involved in dissolution of membrane contacts that are associated with autophagosome formation. Furthermore, little is known about the role of VPS13D in associating autophagy-bound cargo with the site of autophagosome formation, despite having links to the core autophagy machinery. I hypothesize that VPS13D facilitates context-dependent autophagy by associating ubiquitinated cargo with the autophagic machinery and disassembling membrane contact sites at the phagosome assembly site (PAS). Here I propose to determine if VPS13D functions as an autophagy receptor for ubiquitinated cargo and determine the relationship between VPS13D and membrane contact modulator Vacuole Membrane Protein 1 (VMP1). The association of VPS13D and mutations in other factors that regulate autophagic cargo recruitment with human disorders illustrates the importance of studying VPS13D function.

Therapy resistance is a major barrier to long term remission in pediatric T-cell acute lymphoblastic leukemia (T-ALL). The prognosis for children with relapsed or refractory disease is dismal. Leukemia-initiating cells (L-ICs) regenerate disease upon transplantation into mice. They also recapitulate the immunophenotypic complexity of the parent leukemia supporting that, as in normal hematopoiesis, there is a cellular hierarchy among leukemic cells. Our laboratory has previously demonstrated that the L-IC is a committed thymocyte progenitor and resides in the leukemic DN3 population, however, only a fraction of DN3 cells can give rise to disease. L-ICs rely on NOTCH1-induced MYC signaling for survival. Recent studies identified dormant, therapy resistant L-ICs in both murine models and T-ALL patient samples. The role of cell cycle restriction in L-IC latency is incompletely understood. In an effort to uncover pathways that govern L-IC function, we performed single cell RNA sequencing on thymocytes at varying stages of T-cell leukemogenesis using our transgenic Tal1/Lmo2 model. This approach identified a dormant DN3 cluster, marked by low Ki67 expression, which is observed in other murine T-ALL samples. Dormant DN3 cells exhibit high Notch1, but low Myc expression. The transcriptional signature of these cells shows enrichment of genes previously implicated in leukemia initiation or leukemia stem cell function. Dormant DN3 cells show enrichment of the non-canonical Wnt receptor Ryk, which is reported to maintain hematopoietic stem cell self-renewal by limiting proliferation and promoting quiescence. RYK is overexpressed in primary pediatric T-ALL and in Tal1/Lmo2-induced murine T-ALL compared to healthy thymus. This indicates that RYK may not be restricted to this rare subpopulation and moreover, there may be a therapeutic window for RYK inhibition in relapsed T-ALL. The central hypothesis of this proposal is that dormant DN3 cells are quiescent L-ICs that retain proliferative and differentiative capacity, which permits their therapy tolerance and subsequent expansion during relapse. This proposal will identify a gene signature of dormant DN3 cells and uncover the potential role of these cells in T-ALL relapse by evaluating their L-IC function and chemoresistance (Aim 1). Aim 2 will define the non-canonical WNT/RYK signaling network in T-ALL and uncover the role of these pathways in dormant DN3 cells and L-IC function by testing whether inhibition of RYK reduces the L-IC frequency of murine and patient T-ALL cells. Collectively, these studies will provide critical insight to TALL heterogeneity and will lay the foundation for development of L-IC targeted therapy for relapsed disease.

The mammary epithelium is composed of diverse cell populations that are responsible for regulating key processes in mammary gland development, especially lactation. Alveolar progenitor (AP) cells are partially differentiated stem cells that produce alveolar/milk-producing cells and these cells have recently been implicated as cells of origin in breast cancer. Through analysis of published single cell RNA-seq data, integrin β4, a protein known to function primarily in basal mammary epithelial cells to maintain structural integrity of the gland, was found to be expressed in the AP population. This unexpected result suggests that β4 plays a novel role in the AP population and may regulate dynamic processes within the developing mammary gland. This goal of this proposal is to understand the functional role of β4 in the AP cells of the nulliparous and lactating mammary gland and elucidate the mechanism by which β4 regulates this population. Preliminary studies have revealed that β4 expression is necessary to promote progenitor potential of the AP cells and identified LacdiNAc, a novel glycosylation modification, on β4 that we hypothesize is essential for its localization in lipid rafts that promotes its function in the AP population. Using an AP cell culture model as well as a Cre-lox mouse model to conditionally knock out β4 from the AP population, the function of β4 in the AP population will be assessed in vitro and in the virgin and lactating mammary gland. Also, glycomics analyses and biochemical approaches will identify novel glycans on β4 and help in understanding how LacdiNAc affects function of β4 in the AP population. This approach will further our understanding of the novel role of β4 in the AP population and a new mechanism involving LacdiNAc-β4 localization to lipid rafts to regulate alveolar differentiation. These studies have to potential to define novel mechanisms that regulate the AP population during normal mammary gland development as well as breast cancer progression.

Hepatoblastoma (HB), the most common pediatric primary liver tumor, affects children from infancy to five years of age. Surgical resection with adjuvant chemotherapy has saved many young lives. However, the five-year survival rate remains at 70%, and is worse for children with unresectable tumors. Meeting the clinical need for HB-targeted therapies requires a better understanding of how HB tumors are formed and maintained. The transcriptional co-regulator YAP1 is hyper-activated in 79% of HB cases, and recent studies suggest that YAP1 and the Wnt/β-catenin pathway act together to initiate HB tumors. But is YAP1 required to maintain HB tumorigenesis? Preliminary studies using a conditional mouse model of HB—driven by doxycycline-inducible hyperactive YAP1S127A and constitutively active β-catenin—suggest that YAP1 is essential for tumor maintenance. In the presence of doxycycline, YAP1 is expressed, and mice develop HB tumors; withdrawing doxycycline turns off YAP1, resulting in >90% tumor regression within 10 weeks. Transcriptional analyses revealed that hepatocyte differentiation factors and liver metabolic genes were induced in regressing tumors.

Breast cancer is the most common cancer diagnosis in women and is also one of the leading causes of cancer-related mortality in women who suffer from this condition. Tumors that originate in the breast consist of heterogeneous populations of cells. Breast cancer stem cells (BSCSs) are a subtype of tumor cells that have properties similar to normal, tissue stem cells such as the ability to divide slowly and give rise to differentiated cellular lineages. Furthermore, BCSCs have been implicated in tumor initiation, therapy resistance and metastasis to distant organs. Given this information, a greater understanding of the biological processes that sustain BCSCs will lead to the development of novel agents directed against this chemo- and radio-resistant population of tumor cells. The proposed work will explore the role of the α6 integrin splicing variants, α6Aβ1 and α6Bβ1, in the genesis of BCSCs, specifically addressing the mechanism of activation of the TAZ transcriptional coactivator. Integrins are a family of cell surface receptors that function in signal transduction and adhesion to the extracellular matrix. The α6Aβ1 integrin variant is expressed in differentiated, epithelial breast cancer cells and inhibits the acquisition of stem cell properties Conversely, the α6Bβ1 integrin variant is expressed in BCSCs and promotes tumor initiation by activating the Hippo signaling pathway transducer TAZ. TAZ has previously been shown to be important in the functioning of BCSCs, but the mechanism is unknown. Therefore, elucidating the relationship between the α6 integrin splicing variants and TAZ activation will provide insight into mechanisms of breast cancer progression. This proposal will use a cellular and molecular biology approach to establish the mechanism by which TAZ is suppressed in the α6Aβ1 expressing non-stem breast cancer cell population (Aim 1). Biochemical studies will also be undertaken with the purpose of connecting mechanisms of TAZ inactivation with classical Hippo pathway signaling in the non-stem breast cancer cell population (Aim 2). In summary, the studies included in this proposal will increase the understanding of integrin regulation of BCSCs. Our results can provide rationale in designing future targeted therapies for treatment resistant subtypes of breast cancer.

The goal of this proposal is to understand how the Beclin 1/vacuolar protein sorting-associated protein 34 (VPS34) complex contributes to breast cancer progression and to determine how to sensitize tumor cells that are deficient in Beclin 1/VPS34 function to drugs that promote tumor cell death. Breast cancer is the most commonly diagnosed cancer among women worldwide and the second leading cause of cancer related mortality. Despite the availability of targeted therapeutics, the overall survival rate for stage I metastatic disease remains 23%. Therefore, there is a need to develop novel approaches for the treatment of advanced stage breast cancer. The applicant hypothesizes that one such approach could exploit Beclin 1/VPS34 function in breast cancer progression. Beclin 1 is monoallelically deleted in 40% of human breast cancer and there is an inverse correlation between Beclin 1 expression and poor prognosis in ER negative subtypes of breast cancer. In addition, low Beclin 1 expression serves as an independent predictor of patient survival. Beclin 1 interacts with and activates VPS34, the mammalian Class III phosphatidylinositol, to regulate multiple membrane trafficking pathways including autophagy, growth factor receptor trafficking and cytokinesis. The individual contribution of each of these trafficking pathways to cancer is unknown and needs to be investigated to understand the impact of Beclin 1 loss on breast cancer progression. Previous studies in the applicant's lab have shown that loss of Beclin 1 expression is associated with a sustained increase in AKT and ERK signaling downstream of the IGF and EGF receptors. In addition, loss of Beclin 1 expression in breast carcinoma cells leads to increased invasion. These preliminary findings suggest that VPS34 inhibitors, which are currently in clinical trials, may negatively impact tumor treatment. The work that the applicant proposes here will explore how the Beclin 1/VPS34 complex contributes to breast cancer progression. The applicant hypothesizes that inhibiting the Beclin1/VPS34 complex will lead to the upregulation of specific pathways that promote tumor growth and progression and that targeting these pathways in tumors with low Beclin 1 expression or in combination with VPS34 inhibition will suppress tumor cell viability. The goal with this proposal is to dissect out the roe of each Beclin 1/VPS34 complex in breast cancer progression with respect to tumor growth, metastasis, and tumor metabolism both in vitro and in vivo (Aim 1). Furthermore, mechanisms that sensitize breast cancer cells to death upon Beclin 1/VPS34 inhibition will be identified as a means to target Beclin 1 deficient tumors and optimize VPS34 inhibitors as potential therapeutic agents (Aim 2). The studies in this proposal will enhance our understanding of the role of Beclin 1 in breast cancer and can give insight into novel therapies for advanced stage breast cancer disease.

Melanoma is the leading cause of skin cancer death in the United States, with the 5-year survival rate of 20% for patients with advanced disease. Despite improvements in therapy, many patients receive minimal survival benefit and often develop resistance to standard-of-care therapies. Furthermore, a population of patients exist who do not have the appropriate mutations or tumor characteristics to be eligible for new targeted and immunotherapies. In order to provide adequate treatment options for patients who develop resistance or are ineligible for current cutting-edge therapies, new therapeutic targets must be identified. Our lab has discovered a novel melanoma oncogene, growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that promotes melanoma by regulating expression of specific neural crest factors, which has the dual effect of preventing differentiation and suppressing apoptosis9. In addition to these specific factors, we found GDF6 more broadly promotes a gene expression signature that mimics that of the embryonic neural crest. Neural crest identity has previously been identified as a key feature involved in melanoma initiation, progression, and therapeutic resistance. Our studies show knockdown of GDF6 suppresses expression of many of these neural crest genes. Taken together, these data indicate GDF6 is an optimal target for melanoma therapy. As a secreted extracellular protein, GDF6 is amenable to targeting by antibodies. We produced a panel of monoclonal antibodies to target the C-terminal binding region of GDF6 and developed multiple in vitro and in vivo assays to assess candidate antibodies with the most potent action against GDF6 and evaluate their effectiveness as potential melanoma therapeutics. I hypothesize that a subset of antibodies will effectively block GDF6 activity leading to increase differentiation and cell death of melanoma cells in vitro and in vivo. I further hypothesize that blocking GDF6 will suppress neural crest identity in melanoma cells, leading to less aggressive tumor cell characteristics and sensitizing previously resistant cells to standard-of-care therapy. I will evaluate a pre- screened panel of 42 monoclonal antibodies for in vitro and in vivo activity against GDF6 in melanoma cells to identify top candidates with the most potent activity, in parallel with characterizing pharmacokinetic and dynamic properties of the antibodies in vivo. I will further characterize the effects of GDF6 inhibition by these antibodies on neural crest expression profiles and key features of advanced melanoma such as therapeutic resistance, invasiveness, and anchorage independent growth. Additionally, I will analyze potential combinatorial therapies in vitro and in vivo to assess changes in pathway activity for known therapeutic resistance mechanisms. Results of this study will identify lead candidate anti-GDF6 antibodies for first-in-human (FIH) studies and provide appropriate pre-clinical safety data for submission of an FIH application. Furthermore, these data will provide broad insight into the tumorigenic features that are connected to neural crest identity and the result of reversing neural crest characteristics in established melanomas.

In many types of cancer, less differentiated tumors are more strongly associated with a poor patient prognosis. These tumors tend to be more aggressive: they have higher proliferation rates, a greater propensity for invasion and metastasis, and increased resistance to therapy compared to more differentiated tumors. Less differentiated tumors, by their nature, share characteristics with their embryonic cells of origin. In melanoma, these less differentiated tumors are associated with a neural crest identity that is acquired during early stages of tumor initiation and is present through tumor progression. Previous studies have shown that acquisition of a neural crest identity is a necessary step during initiation of early melanoma lesions and supports fundamental properties of aggressive tumors such as invasion and metastasis. However, the mechanisms of generating a neural crest identity are unknown. Recently, our lab has identified growth differentiation factor 6 (GDF6) as a novel melanoma oncogene. GDF6, a bone morphogenetic protein (BMP) ligand, is recurrently amplified in both human and zebrafish melanomas, and expressed in tumors but not normal melanocytes. We have shown GDF6 acts to prevent differentiation and suppress apoptosis in established melanomas, both in vitro and in vivo. Additionally, we have found that GDF6 regulates expression of multiple neural crest and melanocyte factors previously implicated in melanoma. Upon knockdown of GDF6, we observed downregulation of select neural crest factors coupled with upregulation of melanocyte differentiation factors, leading to melanoma cell differentiation and ultimately cell death. These results suggest that GDF6 plays a role in regulating oncogenic neural crest identity. Here, we look to identify the role of GDF6 and BMP signaling in establishing a neural crest identity during melanoma initiation and explore oncogenic characteristics imparted by GDF6 during melanoma progression. I hypothesize that GDF6-depenent BMP signaling acts to initiate a neural crest identity in melanomas to promote tumor initiation and aggressive tumor characteristics. I further hypothesize that loss of BMP activity leads to differentiation (and in tumors death of differentiating cells), making GDF6 an attractive target for differentiation therapy in melanoma.

Breast Cancer is the most frequent malignancy diagnosed in women in the United States and the second leading cause of cancer related mortality in women. Metastasis to distant organs continues to be the greatest obstacle to eradication of this malignancy. Greater understanding of the biological processes that contribute to tumor progression will lead to development of effective therapies against this disease. The insulin receptor substrate (IRS) proteins are important signaling intermediates downstream of the Insulin-like growth factor (IGF-1) receptor and they play a crucial role in the response of tumor cells to IGF-1 stimulation. The two IRS proteins expressed in mammary epithelial cells, IRS1 and IRS2, play different roles in breast cancer. Specifically, tumors that express IRS2 are highly metastatic in comparison to IRS-1 expressing tumors. In addition, IRS2 staining at the membrane in patient tumor samples correlates with decreased overall survival. Studies from our group have identified an IRS2-specific interaction with the microtubule cytoskeleton and demonstrated that disruption of microtubules leads to a decrease in PI3K/Akt activation downstream of IRS2. These findings in conjunction with our preliminary data suggest that the subcellular localization of the IRS proteins plays an important role in their cellular functions. Te work the applicant proposes will explore the potential role of IRS2-microtubule interactions in breast cancer progression, specifically addressing the requirement of this interaction for invasion, glycolysis, PI3K/Akt signaling and tumor metastasis. Our goal with this proposal is to take a molecular biology approach to elucidate the importance of IRS2-microtubule interactions in IRS2 mediated functions (Aim 1). Furthermore, we will use animal models to study the significance of this interaction for tumor progression to metastasis and the impact of IRS2 function on tumor response to IGF-1 receptor inhibitors (Aim 2). The studies proposed in this application will enhance our understanding of the importance of IRS2 in breast cancer progression. Additionally, our results can provide the rationale for the development of novel therapeutic approaches for the treatment of IRS2-dependent malignancies.