Selenoprotein H (SELENOH) is a nucleolar oxidoreductase with DNA binding properties whose function is not well understood. To determine the functional and physiological roles of SELENOH, a knockout of SELENOH was generated in cell lines using CRISPR/Cas9-mediated genomic deletion and in mice by targeted disruption. Based on the sequenced genome, the results of deduced protein sequences indicated various forms of mutants in the CRISPR/Cas9-mediated knockout, including a frame-shift by aberrant splicing and truncated SELENOH by early termination of the translation process. Loss of SELENOH in HeLa cells induced slow cell proliferation, the formation of giant multinucleated cells, accumulation of unrepaired DNA damage and oxidative stress, and cellular senescence. SELENOH cells were enlarged and possessed a single large nucleolus. Atomic force microscope showed increased stiffness in the nucleoli of SELENOH knockout cells, which suggests that SELENOH maintains the flexible structure of the nucleolus. Furthermore, the knockout of SELENOH led to a large-scale reorganization of the nucleolar architecture with the movement of nucleolar protein into nucleolar cap regions in response to oxidative stress. The nucleolar reorganization is dependent on ATM signaling. Altogether, results suggest that SELENOH appears to be a sensor of oxidative stress that plays critical roles in redox regulation and genome maintenance within the nucleolus. To determine the physiological role of SELENOH in vivo, Selenoh knockout mice were generated by targeted deletion through homologous recombination. Selenoh+/− mice were fertile and phenotypically indistinguishable from wild-type littermates. Results from matings of Selenoh+/− mice showed a significantly reduced fraction of Selenoh−/− offspring on the basis of Mendelian segregation. Since some Selenoh−/− were born, it is likely that Selenoh is a partially essential gene in mice. Live-born Selenoh−/− mice were viable and born without apparent phenotypes. Selenoh−/− mice at 2-month of age showed increased GPX activity in the lung but not in the brain and liver. Furthermore, loss of Selenoh resulted in the aggravated formation of aberrant crypt foci in the colon of Selenoh+/− mice that were injected with azoxymethane. Altogether, SELENOH has critical roles in embryogenesis and colorectal carcinogenesis.

No description available.

R-loop is a specific nucleic acid structure, and that forms during RNA transcription. It comprises an RNA:DNA hybrid and displaced ssDNA. R-loops are prevalent and dynamic in the mammalian genome occupying up to 5%. R-loops are known to act as modulators of genome dynamics. They regulate a variety of gene transcription mechanisms and influence genomic stability. Dysregulation of R-loop is linked to a variety of human diseases. For this reason, protein factors involved in DNA and RNA metabolism are known to mediate R-loop resolution. Dysfunction of these factors resulted in aberrant R-loop accumulation, often resulting in transcription disruption and genomic instability as reported in tumorigenesis and a number of genetic disorders, including trinucleotide repeat-associated diseases and neurological disorders. Here I describe the roles of Senataxin (SETX) and Replication protein A (RPA) complex in nucleolar R-loop resolution. I demonstrate their function in nucleolar homeostasis and in particular in RNA polymerase I mediated rRNA transcription and the maintenance of nucleolar structure.This dissertation is composed of three sessions. Section 1: I review nucleolar transcription that generate a complex network of RNAs, and their contribution to nucleolar R-loops formation, with multiple roles in maintaining nucleolar homeostasis. Section 2: I describe novel roles of Replication Protein A in nucleolar homeostasis. Senataxin (SETX) mutations are linked to two different neurological disease: Amyotrophic Lateral Sclerosis (ALS4) and Ataxia Oculomotor Apraxia (AOA2) both defective in R-loop resolution. We show that loss of SETX promotes RPA translocation into nucleolus in an R-loop dependent manner where it associates with rDNA. The same nucleolar RPA phenotype is evident in SETX- deficient AOA2 patient cells. We further explored this phenotype under conditions of CPT- induced genotoxic stress, which is coupled with accumulation of nucleolar R loops. Additionally, we show that loss of RPA decreased 47S pre-rRNA levels, but increased “promoter and pre-rRNA antisense” RNA (PAPAS) and promoter RNA (pRNA). Meanwhile, we also showed loss of RPA disrupted nucleolar structure. Section 3: I describe the identification and characterization of nucleolar lncRNA (PAPAS) which encodes a short polypeptide RIEP that plays a role in combating genomic instability and mitochondrial stress. We show that this novel peptide encoded by PAPAS is localized to the nucleolus and mitochondria but is translocated to the peri-nucleolus and peri-nucleus region upon heat shock induced cellular stresses. We also showed that RIEP facilitates SETX protein stability and plays a role in restricting genomic instability possibly through its association with H3K9me3 which maintains a heterochromatin state. Finally, we show that RIEP interacts with CHCHD2 and C1QBP(P32) and may consequently function in mitochondrial stress responses.

Multiple Myeloma is cancer of plasma cells and is known to be highly invasive. Multiple Myeloma makes up 1% of cancer diagnosis in western countries and affects men more predominantly than women. The American Cancer Association estimates that 32,110 new cases will be diagnosed in the United States in 2019. Lenalidomide is one of the main therapies used for multiple myeloma patients, but it has toxic side effects such as thrombocytopenia, neutropenia, and anemia. The purpose of the study is to investigate new cytotoxic agents for the treatment of multiple myeloma. In addition to lenalidomide alone, this study examined the effects of doxycycline alone and in combination with lenalidomide. Lenalidomide cell cultures were treated at concentrations from 0.5μM to 10μM on untreated 24 well plates and doxycycline concentration ranging from 10μM-80μM. Following incubation, cell viability was tested using MTT assay and the samples were analyzed using spectrophotometry. When compared to lenalidomide, doxycycline monotherapy showed a greater decrease in overall cell viability in preliminary results. Our results show that there is benefit of using 10μM of Doxycycline at higher concentration of 5μM and 10μM of lenalidomide. The potential decrease in the concentration of lenalidomide used by adding doxycycline, may reduce the toxic side effects of lenalidomide. Further studies are necessary to confirm these preliminary results and investigate the mechanism of action in order to determine optimal combinations of these drugs.

Considered the most common malignancy in women in the United States and second leading cause of cancer death among women, breast cancer has had a shift in paradigm of treatment within the recent years and undertaken significant research for new targeted treatment with a more molecular driven approach which has led to increased survival rates amongst women diagnosed with early stage breast cancer. The risk of recurrence however persists overtime, particularly in hormone receptor positive breast cancer, which has demonstrated recurrence rates as late as 30 years after initial diagnosis. This leads to a higher need for increased awareness of late recurrence rates in early stage breast cancer patients and reminds us to be wary of any new findings that in other patients may be considered as benign. We present a case of a 67-year-old female with remote history of locally advanced hormone positive breast cancer in 2005 who underwent mastectomy with lymph node dissection followed by adjuvant chemotherapy, radiation, and endocrine therapy for at least 8 years who presented to our clinic 18 years after initial diagnosis with an enlarging nodule in her neck. Patient underwent a thyroid ultrasound which showed a suspicious thyroid nodule concerning for malignancy classified as TIRADS-5. Further systemic imaging via PET-Scan demonstrated surrounding cervical lymphadenopathy adjacent to the thyroid nodule with increased fluorodeoxyglucose (FDG) avidity. She proceeded to undergo a thyroid fine needle biopsy, which was suspicious for malignancy. A repeat thyroid fine needle biopsy was obtained this time confirming metastatic breast cancer. Considering the rarity of such event, we proceeded with further testing of biopsied tissue via cancer type ID, which confirmed presence of metastatic breast cancer in the thyroid. Patient was informed of now metastatic breast cancer diagnosis with plans to start Faslodex and Ibrance. Unfortunately, she developed rapid disease progression with hospitalization due to a recurrent malignant pericardial effusion suggestive of visceral crisis requiring initiation on palliative chemotherapy with Carboplatin and Gemcitabine. Patient has been tolerating systemic chemotherapy well with interval clinical decrease of more than 50% in size of her surrounding cervical lymphadenopathy and resolution of pericardial effusion. The incidence of thyroid metastatic disease from breast cancer is very rare accounting for only 0.2% of fine needle biopsy aspirations. The most common sites of breast cancer metastasis include lung, bone, liver, and brain. On the other hand, the most common primary malignancies that can cause metastasis to the thyroid are kidney, gastrointestinal tract, and lung. However, as of 2018 around 42 cases of metastatic breast cancer found in the thyroid had been reported and it was also noted that metastatic thyroid involvement of breast cancer could be associated with a poor prognosis. This case represents the importance of being aware of the risk of late recurrence in hormone positive breast cancers, which in turn should result in a lower threshold for thorough workup of common clinical findings in these patients.

In 2019, cancer was the second leading cause of death in the United States. Colorectal cancer is the second most common cancer affecting both men and women. Colorectal screenings are an important preventive health service, with approximately half of cases detected through screening, improving life expectancy among those diagnosed. Previous research has noted differences in screening rates between racial or ethnic groups, with whites being screened at a higher rate than other racial and ethnic groups. Additionally, the prevalence of colorectal cancer is becoming more common among persons younger than 50 years old, prompting testing guidelines to be revised in 2018 that call for testing to begin at 45 instead of age 50. The purpose of this analysis is to investigate this issue further by examining differences in colorectal screening among various racial and ethnic groups. A cross-sectional study using the 2021 Behavioral Risk Factor Surveillance System for respondents from West Virginia and Oregon (combined) was used to examine colon cancer screening by various racial and ethnic populations. Individuals responding receipt of a colonoscopy or sigmoidoscopy were the primary outcome of interest. Racial and ethnic groups include whites-only, blacks-only, other races, multi-racial and those of Hispanic ethnicity. Additional covariates of interest were included in the analysis based on Andersen’s behavior model for health service utilization and includes predisposing (gender, age, education, marital status) enabling (insurance, employment status) need (body mass index, other types of cancers) factors. Bivariate and multivariate logistic regression analysis was used to examine these relationships. The study population included 2,831 adults who self-reported being screened for colon cancer. Overall 61% of white populations reported receipt of colon cancer screening compared to 54% of Black, 52% of Other, and 43% of Hispanic populations (p<0.00). Adjusting for additional covariates of interest, there were no significant differences between colon cancer screening among black populations compared to their white counterparts (aOR=.81; 95% CI=0.40-1.64). Age and education independently predicted being screened for colon cancer. Graduating from college increased an individual’s odds of being screened for colon cancer (OR= 2.1, 95% CI: 1.1-4.0). The odds also increased for individuals between the age of 55 to 64 (OR=4.2, 95% CI: 3.2-5.3) and ages 65 to 74 (OR=10.0, 95% CI: 6.8-14.8). Our study did not find significant differences in colon cancer screening by race/ethnicity in these two states. It is possible the racial and ethnic composition of the states contributed to the observed findings. West Virginia and Oregon are predominantly white. Smaller subpopulation groups within the national BRFSS dataset may not be sufficient to detect important differences in screenings. Furthermore, it is possible that several factors associated with screening (age, education) may be associated with race/ethnicity but are stronger predictors than race/ethnicity itself. Regular screening for colorectal cancer can help with detecting and treating colon cancer. Additional efforts to increase the general knowledge of colorectal cancer risks should be encouraged for individuals who did not graduate from college and are between 45 to 55 years old.

Fibrillary glomerulonephritis (FGN) is a rare disease identified in less than one percent of native kidney biopsy. FGN is characterized by hematuria, edema, renal insufficiency, and high-grade proteinuria. Renal biopsy results typically demonstrate deposition of randomly-arranged fibrils within the capillary wall or mesangium. Positive staining with DnaJ Heat Shock Protein Family Member B9 (DNAJB9) is considered diagnostic. Associations include malignancy, hepatitis C, dysproteinemia, autoimmune disorders, and diabetes mellitus. To our knowledge, this is the first case demonstrating association between fibrillary glomerulonephritis and vulvar squamous cell carcinoma. A 66-year-old year old Caucasian female presented to the emergency department for worsening renal injury. She was diagnosed with vulvar squamous cell carcinoma 5 years prior and underwent radical excision with inguinal lymphadenectomy and CO2 laser treatment. Over the years, she had multiple relapses and received wide local excision and adjuvant radiation, with the last treatment involving radiation being 2.5 years before admission. The patient had a recently identified non-malignant vulvar lesion at the time of admission, which was found to be lichen sclerosus et atrophicus. She was found to have renal dysfunction and nephrotic range proteinuria; her creatinine was 2.84 mg/dL (normal range 0.60–1.10 mg/dL), BUN 33 mg/dL (normal range 6–20 mg/dL), urine protein:creatinine ratio 3.9 mg/g (normal range < 0.15 mg/g). She was started on pulse dose methylprednisolone of 500 mg daily, but her creatinine worsened, necessitating renal biopsy. Renal biopsy findings indicated mesangial expansion and randomly-arranged non-branching fibril deposition. Glomerular immunofluorescence indicated positive staining for IgA, IgG, and DNAJB9. These findings confirmed the diagnosis of fibrillary glomerulonephritis. Screening for associations – coexistent malignancies, hepatitis C, multiple myeloma, and autoimmune disorders – was negative. The patient was started on rituximab and prednisone therapy after confirming the absence of underlying infection. One month after initial hospitalization, she was re-hospitalized for worsening kidney function and required initiation of dialysis, on which she remains dependent. FGN is rapidly progressing and results in end-stage-renal-disease within two years in 50% of individuals. It should be considered as a differential diagnosis in patients with a history of malignancy, especially vulvar squamous cell carcinoma. There is no definitive treatment for FGN. Rituximab can be used in conjunction with steroid therapy, but further research is needed to determine the proper treatment for FGN at various stages of disease manifestation. The original case is published in Cureus, and permission has been received to present this case.

Immune evasion is an emerging hallmark of cancer. Dysregulation of the major histocompatibility complex class I (MHC-I) is a frequent mechanism of immune evasion utilized by tumor cells and is particularly relevant to the pathogenesis of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). A better understanding of MHC-I dysregulation in B-cell lymphomas is necessary to identify factors related to the risk, development, and progression of these tumors. In this thesis, we investigate the role of MHC-I dysregulation in DLBCL and cHL through the application of computational approaches to study genomic data. First, we introduce some background information about the normal function of MHC-I in the immune response to cancer and viral infection as well as the phenomenon of MHC-I dysregulation in the context of cancer. We provide an overview of how factors such as germline zygosity of HLA class I (HLA-I) genes and somatic alterations in the genes B2M and HLA-I that encode the protein subunits of MHC-I contribute to the development of DLBCL and cHL. Second, we present a study of the effects of HLA-I allele zygosity on survival in a cohort of 519 DLBCL patients treated with R-CHOP immunochemotherapy stratified by molecular subtype. Homozygosity in HLA-I was associated with a worse overall survival in patients whose tumors were classified in the “EZB” subtype, associated with somatic mutation in the epigenetic regulator EZH2. We find an association between the zygosity of the genes HLA-B and -C specifically and overall survival in EZB-DLBCL. These findings indicate that HLA-I zygosity may be a risk factor for worse clinical prognosis in patients with the EZB subtype of DLBCL. Third, we present a study of the genetic landscape of cHL tumors that are associated with infection with Epstein-Barr virus (EBV). We analyze inherited HLA-I allele types, somatic mutations, copy number changes, and mutational signatures in a cohort of 57 cHL patients (15 EBV-positive). We find that EBV-positive cHL is genetically distinct from EBV-negative cHL and is characterized by lower somatic mutation load and different activities of mutation signatures. Further, we find that cHL tumors are characterized by different patterns of MHC-I dysregulation depending on the EBV infection status. Germline homozygosity in HLA-I was associated with the EBV-positive subtype of cHL, while somatic alterations in HLA-I were associated with the EBV-negative subtype of cHL. These results suggest that inherited HLA-I homozygosity may be a risk factor for the EBV-positive subtype of cHL. Fourth, we expand our study of HLA-I in virus-associated cHL to perform a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures, recurrent mutations in the RNA helicases DDX3X and EIF4A1, and a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. We find that germline homozygosity in HLA-I is a potential risk factor for the development of EBV-positive cHL, but not other common virus-associated solid or hematological malignancies. Finally, we present in the Appendix a study of the genomic characterization of plasmablastic lymphoma (PBL), a rare EBV-associated B-cell lymphoma that occurs in the context of immunodeficiency caused by human immunodeficiency virus (HIV) infection. We find that PBL is characterized by mutations leading to constitutive activation of the JAK-STAT pathway. We additionally identify recurrent mutations in immune-related genes, such as B2M. These findings indicate a potential role for MHC-I and immune dysregulation in the pathogenesis of other B-cell lymphomas.

No description available.

No description available.