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Tumor-associated inflammation and chromosomal aberrations can play crucial roles in cancer development and progression. In neuroblastoma (NB), the enzyme cyclooxygenase-2 (COX-2) is associated with copy number alterations on the long arm of chromosome 11 (Ch 11q), defining an aggressive disease subset. This retrospective study included formalin-fixed paraffin-embedded tumor samples collected from nine patients during diagnosis at the pediatric Pequeno Principe Hospital, Curitiba, PR, Brazil, and post-chemotherapy (CT). COX-2 expression was evaluated using immunohistochemistry and correlated with the genome profile of paired pre- and post-CT samples, determined by array comparative genomic hybridization. A systems biology approach elucidated the

One-third of all estrogen receptor (ER)–positive breast tumors treated with endocrine therapy fail to respond, and the remainder is likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers each year and, whereas the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of ERα and increased expression of the estrogen-related receptor γ (ERRγ). Knockdown of ERRγ in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRγ blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRγ-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRγ/AP1 signaling in the development of TAM resistance and suggest that expression of ERRγ may be a marker of poor TAM response. [Cancer Res 2008;68(21):8908–17]

Background Embryonic stem cell markers, such as SOX2, NANOG, and OCT4, are transcription factors expressed in pluripotent stem cells, involved in the mediation of pluripotency and self-renewal. Especially after the discovery of cancer stem cells, these proteins have been associated with several types of neoplasia, including astrocytomas. In the pediatric population, astrocytomas are the most common solid neoplasia and present the highest mortality rates. Methods Our study evaluated 5 polymorphisms in SOX2, NANOG, and POU5F1 genes in 101 pediatric astrocytoma samples. Results We describe the associations between wild and polymorphic alleles in astrocytomas. Conclusions In our results, the intronic polymorphic G allele in SOX2 rs77677339 [G/A] had a borderline association with low-grade astrocytomas, and the intronic polymorphic T allele in NANOG rs10845877 [C/T] showed a higher frequency in grade 2, compared to grade 1 astrocytomas, thus showing promising results. Impact Our study is relevant because it shows a potential correlation between polymorphic embryonic stem cell marker genes and pediatric astrocytomas.

BACKGROUND: Penile cancer (PeCa) is a rare disease, but its incidence has increased worldwide, mostly in HPV+ patients. Nevertheless, there is still no targeted treatment for this carcinoma. OBJECTIVE: To predict the main signaling pathways involved in penile tumorigenesis and its potential drug targets. METHODS: Genome-wide copy number profiling was performed in 28 PeCa. Integration analysis of CNAs and miRNAs and mRNA targets was performed by DIANA-TarBase v.8. The potential impact of the miRNAs/target genes on biological pathways was assessed by DIANA-miRPath v.3.0. For each miRNA, KEGG pathways were generated based on the tarbase and microT-CDS algorithms. Pharmaco-miR was used to identify associations between miRNAs and their target genes to predict druggable targets. RESULTS: 269 miRNAs and 2,395 genes were mapped in cytobands with CNAs. The comparison of the miRNAs mapped at these cytobands and the miRNAs that were predicted to regulate the genes also mapped in these regions, resulted in a set of common 35 miRNAs and 292 genes. Enrichment pathway revealed their involvement in five top signaling pathways. EGFR and COX2 were identified as potential druggable targets. CONCLUSION: Our data indicate the potential use of EGFR and COX2 inhibitors as a target treatment for PeCa patients.

ObjectiveThe Brazilian state of Paraná has suffered from COVID-19 effects, understanding predictors of increased mortality in health system interventions prevent hospitalisation of patients. We selected the best models to evaluate the association of death with demographic characteristics, symptoms and comorbidities based on three levels of clinical severity for COVID-19: non-hospitalised, hospitalised non-ICU ward and ICU ward.DesignCross-sectional survey using binomial mixed models.SettingCOVID-19-positive cases diagnosed by reverse transcription–PCR of municipalities located in Paraná State.PatientsCases of anonymous datasets of electronic medical records from 1 April 2020 to 31 December 2020.Primary and secondary outcome measuresThe best prediction factors were chosen based on criteria after a stepwise analysis using multicollinearity measure, lower Akaike information criterion and goodness-of-fit χ2tests from univariate to multivariate contexts.ResultsMale sex was associated with increased mortality among non-hospitalised patients (OR 1.76, 95% CI 1.47 to 2.11) and non-ICU patients (OR 1.22, 95% CI 1.05 to 1.43) for symptoms and for comorbidities (OR 1.89, 95% CI 1.59 to 2.25, and OR 1.30, 95% CI 1.11 to 1.52, respectively). Higher mortality occurred in patients older than 35 years in non-hospitalised (for symptoms: OR 4.05, 95% CI 1.55 to 10.54; and for comorbidities: OR 3.00, 95% CI 1.24 to 7.27) and in hospitalised over 40 years (for symptoms: OR 2.72, 95% CI 1.08 to 6.87; and for comorbidities: OR 2.66, 95% CI 1.22 to 5.79). Dyspnoea was associated with increased mortality in non-hospitalised (OR 4.14, 95% CI 3.45 to 4.96), non-ICU (OR 2.41, 95% CI 2.04 to 2.84) and ICU (OR 1.38, 95% CI 1.10 to 1.72) patients. Neurological disorders (OR 2.16, 95% CI 1.35 to 3.46), neoplastic (OR 3.22, 95% CI 1.75 to 5.93) and kidney diseases (OR 2.13, 95% CI 1.36 to 3.35) showed the majority of increased mortality for ICU as well in the three levels of severity jointly with heart disease, diabetes and CPOD.ConclusionsThese findings highlight the importance of the predictor’s assessment for the implementation of public healthcare policy in response to the COVID-19 pandemic, mainly to understand how non-pharmaceutical measures could mitigate the virus impact over the population.

MiR-150-5p is frequently deregulated in cancer, with expression and mode of action varying according to the tumor type. Here, we investigated the expression levels and role of miR-150-5p in the aggressive breast cancer subtype triple-negative breast cancer (TNBC). MiR-150-5p expression levels were analyzed in tissue samples from 113 patients with invasive breast cancer (56 TNBC and 57 non-TNBC) and 41 adjacent non-tumor tissues (ANT). Overexpression of miR-150-5p was observed in tumor tissues compared with ANT tissues and in TNBC compared with non-TNBC tissues. MiR-150-5p expression levels were significantly associated with high tumor grades and the Caucasian ethnicity. Interestingly, high miR-150-5p levels were associated with prolonged overall survival. Manipulation of miR-150-5p expression in TNBC cells modulated cell proliferation, clonogenicity, migration, and drug resistance. Manipulation of miR-150-5p expression also resulted in altered expression of its mRNA targets, including epithelial-to-mesenchymal transition markers, MYB, and members of the SRC pathway. These findings suggest that miR-150-5p is overexpressed in TNBC and contributes to the aggressiveness of TNBC cells in vitro.

The R337H is a TP53 germline pathogenic variant that has been associated with several types of cancers, including breast cancer. Our main objective was to determine the frequency of the R337H variant in sporadic breast cancer patients from Paraná state, South Brazil, its association with prognosis and its impact in genomic instability. The genotyping of 805 breast cancer tissues revealed a genotypic and allelic frequency of the R337H variant of 2.36% and 1.18%, respectively. In these R337H+ cases a lower mean age at diagnosis was observed when compared to the R337H-cases. Array-CGH analysis showed that R337H+ patients presented a higher number of copy number alterations (CNAs), compared to the R337H−. These CNAs affected genes and miRNAs that regulate critical cancer signaling pathways; a number of these genes were associated with survival after querying the KMplot database. Furthermore, homozygous (R337H+/R337H+) fibroblasts presented increased levels of copy number variants when compared to heterozygous or R337H− cells. In conclusion, the R337H variant may contribute to 2.36% of the breast cancer cases without family cancer history in Paraná. Among other mechanisms, R337H increases the level of genomic instability, as evidenced by a higher number of CNAs in the R337H+ cases compared to the R337H−.

This review was conducted to present the main neuroblastoma (NB) clinical characteristics and the most common genetic alterations present in these pediatric tumors, highlighting their impact in tumor cell aggressiveness behavior, including metastatic development and treatment resistance, and patients’ prognosis. The distinct three NB cell lineage phenotypes, S-type, N-type, and I-type, which are characterized by unique cell surface markers and gene expression patterns, are also reviewed. Finally, an overview of the most used NB cell lines currently available for in vitro studies and their unique cellular and molecular characteristics, which should be taken into account for the selection of the most appropriate model for NB pre-clinical studies, is presented. These valuable models can be complemented by the generation of NB reprogrammed tumor cells or organoids, derived directly from patients’ tumor specimens, in the direction toward personalized medicine.

Funda??o de Amparo ? Pesquisa e ao Desenvolvimento Cient?fico e Tecnol?gico do Maranh?o. Grant Numbers: 00971/17, 01352/16. Federal University of Maranh?o. Postgraduate Program in Health Science. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Department of Biology. Laboratory of Genetics and Molecular Biology. S?o Lu?s, MA, Brazil. Aldenora Bello Cancer Hospital. S?o Lu?s, MA, Brazil. Aldenora Bello Cancer Hospital. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Postgraduate Program in Health Science. S?o Lu?s, MA, Brazil / Federal University of Maranh?o. Department of Biology. Laboratory of Genetics and Molecular Biology. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Public Health Department. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Department of Pathology. Biology Undergraduate Course. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Postgraduate Program in Health Science. S?o Lu?s, MA, Brazil. Federal University of Maranh?o. Department of Pathology. Biology Undergraduate Course. S?o Lu?s, MA, Brazil. Federal University of Par?. Oncology Research Center. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecido e citogen?tica. Ananindeua, PA, Brasil. Federal University of Maranh?o. Department of Physiological Sciences. S?o Lu?s, MA, Brazil. Faculdades Pequeno Pr?ncipe. Instituto de Pesquisa Pel? Pequeno Pr?ncipe. Curitiba, PR, Brazil / Georgetown Lombardi Comprehensive Cancer Center. Department of Oncology. Washington, District of Columbia, United States. Federal University of Maranh?o. Department of Biology. Laboratory of Genetics and Molecular Biology. S?o Lu?s, MA, Brazil. The incidence of penile cancer (PeCa) is increasing worldwide, however, the highest rates are reported in underdeveloped countries. The molecular mechanisms that underly the onset and progression of these tumors are still unclear. Therefore, our goal was to determine the genome?wide copy number alterations and the involvement of human papiloma virus (HPV) (TP53 and RB1), inflammatory (COX2 and EGFR), and PI3K/AKT pathway (AKT1, AKT2, EGFR, ERBB3, ERBB4, PIK3CA, and PTEN) associated genes in patients with PeCa from a high incidence region in Brazil (Maranh?o). HPV genotyping was performed by nest?PCR and genome sequencing, copy number alterations (CNAs) by array comparative genomic hybridization and gene copy number status, gene, and protein expression by quantitative polymerase chain reaction, reverse transcriptase?quantitative polymerase chain reaction, and immunohistochemistry, respectively. HPV genotyping revealed one of the highest frequencies of HPV reported in PeCa, affecting 96.4% of the cases. The most common CNAs observed were located at the HPV integration sites, such as 2p12?p11.2 and 14q32.33, where ADAM 6, KIAA0125, LINC00226, LINC00221, and miR7641?2, are mapped. Increased copy number of ERBB3 and EGFR genes were observed in association with COX2 and EGFR overexpression, reinforcing the role of the inflammatory pathway in PeCa, and suggesting anti?EGFR and anti?COX2 inhibitors as promising therapies for PeCa. Additionally, TP53 and RB1 messenger RNA downregulation was observed, suggesting the occurrence of other mechanisms for repression of these oncoproteins, in addition to the canonical HPV/TP53/RB1 signaling pathway. Our data reinforce the role of epigenetic events in abnormal gene expression in HPV?associated carcinomas and suggest the pivotal role of HPV driving CNAs and controlling gene expression in PeCa.

Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

The COX-2 protein, encoded by the PTGS2 gene, is related to tumor progression in adult and pediatric cancer. In neuroblastoma (NB), COX-2 was associated with loss of heterozygosity on the long arm of chromosome 11 (Ch11q loss of heterozygosity, LOH), defining a subset of aggressive disease. The present study aimed to investigate the protein expression of COX-2 in a set of 82 pre-chemotherapy (CT) and 20 post-CT NB specimens and its correlation with clinical and genomic data. A systems biology approach elucidated the network interaction of PTGS2 and other inflammation-related genes with those codified in the Ch11q deleted regions. The results indicated a significantly higher expression of COX-2 in post-CT samples. In addition, a significant positive correlation between the presence of aberrations in Ch11q and COX-2 levels and an indirect connection between the COX-2 gene and extracellular matrix remodeling (ECM)-related proteins were observed. Our findings suggest that deregulation of ECM proteolysis in Ch11q–deleted NB could elicit stromal alterations, triggering inflammatory responses via COX-2 overexpression, ultimately supporting NB progression.

Triple negative breast cancer (TNBC), a clinically aggressive breast cancer subtype, affects 15–35% of women from Latin America. Using an approach of direct integration of copy number and global miRNA profiling data, performed simultaneously in the same tumor specimens, we identified a panel of 17 miRNAs specifically associated with TNBC of ancestrally characterized patients from Latin America, Brazil. This panel was differentially expressed between the TNBC and non-TNBC subtypes studied (p ≤ 0.05, FDR ≤ 0.25), with their expression levels concordant with the patterns of copy number alterations (CNAs), present mostly frequent at 8q21.3-q24.3, 3q24-29, 6p25.3-p12.2, 1q21.1-q44, 5q11.1-q22.1, 11p13-p11.2, 13q12.11-q14.3, 17q24.2-q25.3 and Xp22.33-p11.21. The combined 17 miRNAs presented a high power (AUC = 0.953 (0.78–0.99);95% CI) in discriminating between the TNBC and non-TNBC subtypes of the patients studied. In addition, the expression of 14 and 15 of the 17miRNAs was significantly associated with tumor subtype when adjusted for tumor stage and grade, respectively. In conclusion, the panel of miRNAs identified demonstrated the impact of CNAs in miRNA expression levels and identified miRNA target genes potentially affected by both CNAs and miRNA deregulation. These targets, involved in critical signaling pathways and biological functions associated specifically with the TNBC transcriptome of Latina patients, can provide biological insights into the observed differences in the TNBC clinical outcome among racial/ethnic groups, taking into consideration their genetic ancestry.

Copy number alterations (CNAs) are a frequent feature in human breast cancer, and one of the hallmarks of genomic instability. The FOSL1, GSTP1 and CCND1 genes are located at 11q13, a cytoband commonly affected by CNA in breast cancer, with relevant function in progression and invasion. Our main goal was to analyze CNAs of these genes and determine their association with breast cancer subtypes. Seventy-three cases of invasive breast tumors [52 Luminal, 7 HER2+ and 14 triple negative (TNBC) subtypes] were analyzed by TaqMan assays. CNAs were observed for all genes, with gains more frequently observed. Gains of the FOSL1 gene were observed in 71% of the cases. This gene was the only one with a statistically significant difference (p

MicroRNAs (miRNAs) are critical modulators of endothelial homeostasis, which highlights their involvement in vascular diseases, including those caused by virus infections. Our main objective was to identify miRNAs involved in the endothelial function and determine their expression in post-mortem lung biopsies of COVID-19 patients with severe respiratory injuries and thrombotic events. Based on functional enrichment analysis, miR-26a-5p, miR-29b-3p, and miR-34a-5p were identified as regulators of mRNA targets involved in endothelial and inflammatory signaling pathways, as well as viral diseases. A miRNA/mRNA network, constructed based on protein–protein interactions of the miRNA targets and the inflammatory biomarkers characterized in the patients, revealed a close interconnection of these miRNAs in association to the endothelial activation/dysfunction. Reduced expression levels of selected miRNAs were observed in the lung biopsies of COVID-19 patients ( n = 9) compared to the Controls ( n = 10) ( P < 0.01-0.0001). MiR-26a-5p and miR-29b-3p presented the best power to discriminate these groups (area under the curve (AUC) = 0.8286, and AUC = 0.8125, respectively). The correlation analysis of the miRNAs with inflammatory biomarkers in the COVID-19 patients was significant for miR-26a-5p [IL-6 ( r2 = 0.5414), and ICAM-1 ( r2 = 0.5624)], and miR-29b-3p [IL-4 ( r2 = 0.8332) and IL-8 ( r2 = 0.2654)]. Altogether, these findings demonstrate the relevance and the non-random involvement of miR-26a-5p, miR-29b-3p, and miR-34a-5p in endothelial dysfunction and inflammatory response in patients with SARS-CoV-2 infection and the occurrence of severe lung injury and immunothrombosis.

Background To investigate the global expression profile of miRNAs, their impact on cellular signaling pathways, and their association with poor prognostic parameters in African-American (AA) patients with triple negative breast cancer (TNBC). Methods Twenty-five samples of AA TNBC patients were profiled for global miRNA expression and stratified considering three clinical-pathological parameters: tumor size, lymph node (LN), and recurrence (REC) status. Differential miRNA expression analysis was performed for each parameter, and their discriminatory power was determined by Receiver Operating Characteristic (ROC) curve analysis. KMplotter was assessed to determine the association of the miRNAs with survival, and functional enrichment analysis to determine the main affected pathways and miRNA/mRNA target interactions. Results A panel of eight, 23 and 27 miRNAs were associated with tumor size, LN, and REC status, respectively. Combined ROC analysis of two (miR-2117, and miR-378c), seven (let-7f-5p, miR-1255b-5p, miR-1268b, miR-200c-3p, miR-520d, miR-527, and miR-518a-5p), and three (miR-1200, miR-1249-3p, and miR-1271-3p) miRNAs showed a robust discriminatory power based on tumor size (AUC = 0.917), LN (AUC = 0.945) and REC (AUC = 0.981) status, respectively. Enrichment pathway analysis revealed their involvement in proteoglycans and glycan and cancer-associated pathways. Eight miRNAs with deregulated expressions in patients with large tumor size, positive LN metastasis, and recurrence were significantly associated with lower survival rates. Finally, the construction of miRNA/mRNA networks based in experimentally validated mRNA targets, revealed nodes of critical cancer genes, such as AKT1, BCL2, CDKN1A, EZR and PTEN. Conclusions Altogether, our data indicate that miRNA deregulated expression is a relevant biological factor that can be associated with the poor prognosis in TNBC of AA patients, by conferring to their TNBC cells aggressive phenotypes that are reflected in the clinical characteristics evaluated in this study.

Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the “omics” studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.

Graphical abstract, In this review, we highlight the interaction of SARS-CoV-2 virus and host genomes, reporting the current studies on the sequence analysis of SARS-CoV-2 isolates and host genomes from diverse world populations. The main genetic variants that are present in both the virus and host genomes were particularly focused on the ACE2 and TMPRSS2 genes, and their impact on the patients' susceptibility to the virus infection and severity of the disease. Finally, the interaction of the virus and host non-coding RNAs is described in relation to their regulatory roles in target genes and/or signaling pathways critically associated with SARS-CoV-2 infection. Altogether, these studies provide a significant contribution to the knowledge of SARS-CoV-2 mechanisms of infection and COVID-19 pathogenesis. The described genetic variants and molecular factors involved in host/virus genome interactions have significantly contributed to defining patient risk groups, beyond those based on patient age and comorbidities, and they are promising candidates to be potentially targeted in treatment strategies for COVID-19 and other viral infectious diseases.

Background: Brazil is facing a surge in COVID-19 infection, with heterogeneous regional spread associated with varying infection and death rates across the country’s states. This study aimed to thoroughly analyze the association of death with demographic characteristics, symptoms, and comorbidities at three levels of COVID-19 severity—in non-hospitalized, non-ICU-ward, and ICU-ward patients—in a state-wide context in South Brazil. Methods: We selected 221,414 cases in the state of Parana diagnosed by positive RT-PCR tests, defining outcome based on electronic medical records including demographic characteristics (sex, age, HDI), description of symptoms (114,025 cases with at least one symptom—SY group), and comorbidities (116,229 cases with at least one comorbidity—CM group). Logistic regression was performed to test the association between deaths with demographic characteristics and categorical variables in three levels of medical intervention within the SY and CM groups. Findings: Registered ICU deaths ranged between 68·62% (SY) and 69·6% (CM). Males presented increased mortality among non-hospitalized patients (SY: OR=1·81, 95% CI=1·51–2·17; CM: OR=1.97, 95% CI=1·66–2·34) and non-ICU patients (SY: OR=1·24, 95% CI=1·07–1·45; CM: OR=1·35, 95% CI=1·15–1·57). Lower human development index (HDI) was associated with increased mortality in non-hospitalized patients (SY: OR=0·01, 95% CI=0·02–0·08, CM: OR=0·02, 95% CI=0·01– 0·11). A higher mortality rate occurred in patients older than 40 years (non-hospitalized OR=25·65, 95% CI=8·17–155·36, non-ICU OR=5·05, 95% CI=1·87–20·75, and ICU OR=3·91, 95% CI=1·27–14·58), with strong association in elderly SY and CM adults. The main symptoms associated with increased mortality were dyspnoea in non-hospitalized (OR=3·71, 95% CI=3·10–4·44), non-ICU (OR=2·47, 95% CI=2·11–2·90), and ICU patients (OR=1·43, 95% CI=1·16–1·77). In all patients, diabetes, heart, neurological, and kidney diseases were associated with death (e.g., in non-hospitalized patients: OR=2·12, 95% CI=1·72–2·60; OR=1·84, 95% CI=1·47–2·29; OR=2·18, 95% CI=1·46–3·17 OR=2·00, 95% CI=1·20–3·19, respectively). Interpretation: The demographic characteristics “male sex” and “age > 40 years” were unequivocally associated with death and with lower HDI in non-hospitalized patients. Other categorical variables—dyspnoea, diabetes, heart disease, neurological disease, and kidney disease—were associated with most patients’ mortality. Our study highlights the importance of these predictors for the implementation of public healthcare policy in response to the pandemic. Funding: Araucaria Foundation – FAAP-PR, State Secretariat of Science, Technology and Higher Education – SETI –PR. ABWB currently receives a research productivity scholarship from CNPq (314288/2018-0). Declaration of Interests: We declare no competing interests. Ethics Approval Statement: The study received the ethical approval number CAAE 39108020.0.0000.0107, registered on the Plataforma Brasil – Comissao Nacional de Etica em Pesquisa database (https://plataformabrasil.saude.gov.br).

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).

MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.

Purpose Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, is genetically heterogeneous which challenges the identification of clinically effective molecular makers. Extracellular vesicles (EVs) are key players in the intercellular signaling communication and have been shown to be involved in tumorigenesis. The main goal of this study was to evaluate the role and mechanisms of EVs derived from TNBC cells in modulating proliferation and cytotoxicity to chemotherapeutic agents in non-tumorigenic breast cells (MCF10A). Methods EVs were isolated from TNBC cell lines and characterized by nanoparticle tracking analysis, Western blot, and transmission electron microscopy. MCF10A cells were treated with the isolated EVs and evaluated for cell proliferation and cytotoxicity to Docetaxel and Doxorubicin by the MTT and MTS assays, respectively. Gene and miRNA expression profiling was performed in the treated cells to determine expression changes that may be caused by EVs treatment. Results MCF10A cells treated with HCC1806-EVs (MCF10A/HCC1806-EVs) showed a significant increase in cell proliferation and resistance to the therapeutic agents tested. No significant effects were observed in the MCF10A cells treated with EVs derived from MDA-MB-231 cells. Gene and miRNA expression profiling revealed 138 genes and 70 miRNAs significantly differentially expressed among the MCF10A/HCC1806-EVs and the untreated MCF10A cells, affecting mostly the PI3K/AKT, MAPK, and HIF1A pathways. Conclusion EVs isolated from the HCC1806 TNBC cells are capable of inducing proliferation and drug resistance on the non-tumorigenic MCF10A breast cells, potentially mediated by changes in genes and miRNAs expression associated with cell proliferation, apoptosis, invasion, and migration. Electronic supplementary material The online version of this article (10.1007/s10549-018-4925-5) contains supplementary material, which is available to authorized users.

Introduction: Triple negative breast cancer (TNBC) is a highly aggressive form of breast cancer prevalent in African-American (AA) women defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative. Because ER- and HER2-targeted therapies are ineffective in TNBC, systemic chemotherapy is the standard of care and there is a tremendous need for new effective therapies with less toxicity. Steroid hormone receptors are highly druggable targets, and orphan nuclear receptors, members of the nuclear receptor superfamily, are emerging as targets for cancer therapy. In fact, we have previously shown that treatment of TNBC cells with a small molecule agonist ligand (DY131) for estrogen related receptor beta (ERRβ), has growth inhibitory and anti-mitotic activity. We have also shown that increased mRNA expression of ERRβ, correlates with better recurrence- and distant metastasis-free survival in TNBC/basal-like breast cancer. The goal of our current work is to comprehensively characterize ERRβ copy number and mRNA status in TNBC and determine its association with patients' prognosis. Methods: ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by array-CGH, using the Agilent SurePrint G3 Human CGH platform. ESRRB mRNA data and its association with overall survival was determined in systemically untreated patients from METABRIC using Illumina gene expression array data (probe ID ILMN_1707398). Results: Copy number alterations (CNAs). Copy number losses at the ESRRB locus (14q24.3) were observed in 10/56 (17.8%) of TNBC vs. 10/50 (20%) of nonTNBC, while copy number gains were detected in 43/56 (76.8%) of TNBC vs. 29/50 (58%) of nonTNBC (c2 *p=0.036). Interestingly, in both TNBC and non-TNBC, ESRRB loss was seen with markedly higher frequency in AA patients when compared to Caucasian (CA) patients (c2 *p=0.012 for TNBC, p=0.052 for non-TNBC). mRNA expression. Among patients not treated with systemic chemotherapy in the METABRIC dataset, low ESRRB mRNA was significantly associated with shorter overall survival in TNBC, but not ER+ or HER2+ patients (TNBC hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Low ESRRB also correlated with reduced overall survival in TP53 mutant (but not wild type) tumors (hazard ratio 0.28, 95% confidence interval 0.1-0.82, *p=0.013). Conclusions: ESRRB presents significantly high levels of copy number losses in TNBC when compared to non-TNBC tumors. In breast tumors from AA women, both the TNBC and non-TNBC subtypes are significantly more likely to have reduced ESRRB copy number vs. CA women. Low ESRRB mRNA expression predicts for poor overall survival in TNBC and TP53 mutant tumors. These data advocate that ERRβ expression has prognostic value in breast cancer, particularly TNBC. Future goals include immunohistochemistry staining, and analysis, of a tissue microarray consisting of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+); as well as ERRβ overexpression and knock-down studies in TNBC cell lines to define the role it plays in TNBC. Citation Format: Fernandez AI, Graham G, Győrffy B, Cavalli L, Mahajan A, Riggins RB. ERRβ copy number and expression in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-09.

Triple-negative breast cancer (TNBC) can be further classified into androgen receptor (AR)-positive TNBC and AR-negative TNBC or quadruple-negative breast cancer (QNBC). Here, we investigated genomic instability in 53 clinical cases by array-CGH and miRNA expression profiling. Immunohistochemical analysis revealed that 64% of TNBC samples lacked AR expression. This group of tumors exhibited a higher level of copy number alterations (CNAs) and a higher frequency of cases affected by CNAs than TNBCs. CNAs in genes of the chromosome instability 25 (CIN25) and centrosome amplification (CA) signatures were more frequent in the QNBCs and were similar between the groups, respectively. However, expression levels of CIN25 and CA20 genes were higher in QNBCs. miRNA profiling revealed 184 differentially expressed miRNAs between the groups. Fifteen of these miRNAs were mapped at cytobands with CNAs, of which eight (miR-1204, miR-1265, miR-1267, miR-23c, miR-548ai, miR-567, miR-613, and miR-943), and presented concordance of expression and copy number levels. Pathway enrichment analysis of these miRNAs/mRNAs pairings showed association with genomic instability, cell cycle, and DNA damage response. Furthermore, the combined expression of these eight miRNAs robustly discriminated TNBCs from QNBCs (AUC = 0.946). Altogether, our results suggest a significant loss of AR in TNBC and a profound impact in genomic instability characterized by CNAs and deregulation of miRNA expression.

Background Triple negative breast cancer (TNBC) tumors are estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor-negative. TNBC is responsive to chemotherapy, but chemotherapy might be underused in some patient subgroups. The goal of the present study was to characterize the patterns of chemotherapy use (uptake and completion) in TNBC patients. Patients and Methods Women with primary invasive, nonmetastatic breast cancer were recruited in Washington, DC, and Detroit. Data were collected using a standardized telephone survey that captured sociocultural and health care process factors. Clinical data were abstracted from the medical records. We used χ 2 tests to access the association between the receipt of chemotherapy use (initiation and completion) and categorical variables, and t tests were used for continuous variables. Logistic regression models were used to evaluate the factors associated with chemotherapy uptake. Results Women with TNBC (16% of sample) were more likely to be black than white (68% vs. 32%; P P = .01) and in women without (vs. with) healthcare discrimination (35% vs. 25%; P = .04). In multivariable models, only race was associated with the receipt of chemotherapy. Black women were more likely to receive chemotherapy than were white women. The odds ratio of receiving chemotherapy by race was 4.1 (95% confidence interval, 1.3-13.1). Each 1-year increase in age was associated with a lower likelihood of chemotherapy completion (odds ratio, 0.9; 95% confidence interval, 0.826-0.981; P = .02). Women with at least some college were less likely to complete chemotherapy than were those with other education levels ( P = .02). Conclusion A substantial number of TNBC patients failed to receive and/or complete chemotherapy. Differences in chemotherapy uptake by race and sociocultural factors diminished in multivariable models but age and stage remained significant. Suboptimal treatment among women with TNBC could contribute to adverse outcomes. Future investigations are necessary to assess whether the noninitiation and/or noncompletion of chemotherapy is clinically warranted.

// Bruna Sugita 1 , Mandeep Gill 2 , Akanskha Mahajan 2 , Anju Duttargi 2 , Saurabh Kirolikar 2 , Rodrigo Almeida 1 , Kenny Regis 2 , Olusayo L. Oluwasanmi 2 , Fabio Marchi 3 , Catalin Marian 4, 8 , Kepher Makambi 2, 5 , Bhaskar Kallakury 6 , Laura Sheahan 7 , Iglenir J.Cavalli 1 , Enilze M. Ribeiro 1 , Subha Madhavan 2, 7 , Simina Boca 2, 7 , Yuriy Gusev 2, 7 , Luciane R. Cavalli 2 1 Department of Genetics, Federal University of Parana, Curitiba, PR, Brazil 2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 3 International Research Center-CIPE, A. C. Camargo Cancer Center, Sao Paulo, SP, Brazil 4 The Ohio State University Comprehensive Cancer Center, Division of Cancer Prevention and Control, College of Medicine, The Ohio State University, Columbus, Ohio 5 Departments of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University, Washington, DC USA 6 Department of Pathology, Georgetown University Medical Center, Washington, DC, USA 7 Innovation Center for Biomedical Informatics, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 8 The University of Medicine and Pharmacy Timisoara, Timisoara, Romania Correspondence to: Luciane R. Cavalli, email: lrc@georgetown.edu Keywords: microRNA, triple negative breast cancer, African American, non-Hispanic white, copy number Received: July 02, 2016 Accepted: October 25, 2016 Published: November 02, 2016 ABSTRACT Triple Negative Breast Cancer (TNBC), a clinically aggressive subtype of breast cancer, disproportionately affects African American (AA) women when compared to non-Hispanic Whites (NHW). MiRNAs(miRNAs) play a critical role in these tumors, through the regulation of cancer driver genes. In this study, our goal was to characterize and compare the patterns of miRNA expression in TNBC of AA ( n = 27) and NHW women ( n = 30). A total of 256 miRNAs were differentially expressed between these groups, and distinct from the ones observed in their respective non-TNBC subtypes. Fifty-five of these miRNAs were mapped in cytobands carrying copy number alterations (CNAs); 26 of them presented expression levels concordant with the observed CNAs. Receiving operating characteristic (ROC) analysis showed a good power (AUC ≥ 0.80; 95% CI) for over 65% of the individual miRNAs and a high combined power with superior sensitivity and specificity (AUC = 0.88 (0.78−0.99); 95% CI) of the 26 miRNA panel in discriminating TNBC between these populations. Subsequent miRNA target analysis revealed their involvement in the interconnected PI3K/AKT, MAPK and insulin signaling pathways. Additionally, three miRNAs of this panel were associated with early age at diagnosis. Altogether, these findings indicated that there are different patterns of miRNA expression between TNBC of AA and NHW women and that their mapping in genomic regions with high levels of CNAs is not merely physical, but biologically relevant to the TNBC phenotype. Once validated in distinct cohorts of AA women, this panel can potentially represent their intrinsic TNBC genome signature.

PurposeTriple negative breast cancer (TNBC)/ basal-like breast cancer (BLBC) is a highly aggressive form of breast cancer prevalent in African-American (AA) women. We previously reported that a small molecule agonist ligand for the orphan nuclear receptor estrogen-related receptor beta (ERRβ orESRRB) has growth inhibitory and anti-mitotic activity in TNBC cell lines. In this study, we evaluate the association ofESRRBmRNA, copy number levels, and protein expression with demographic, clinicopathological, and gene expression features in breast tumor clinical specimens.MethodsESRRBmRNA level expression and clinical associations were analyzed using RNAseq data. Array-based comparative genomic hybridization determinedESRRBcopy number in AA and Caucasian women. Transcription factor activity was measured using promoter-reporter luciferase assays in TNBC cell lines. Semi-automatic quantification of immunohistochemistry measured ERRβ protein expression on a 150-patient tissue microarray series.ResultsESRRBmRNA expression is significantly lower in TNBC/BLBC vs. other breast cancer subtypes. There is no evidence ofESRRBcopy number loss.ESRRBmRNA expression is correlated with the expression of genes associated with neuroactive ligand-receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs. TheESRRBmessage is alternatively spliced into three isoforms, which we show have different transcription factor activity in basal-like vs. other TNBC cell lines. We further show that the ERRβ2 and ERRβsf isoforms are broadly expressed in breast tumors at the protein level.ConclusionsDecreasedESRRBmRNA expression, and distinct patterns of ERRβ isoform subcellular localization and transcription factor activity are key features in TNBC/BLBC.

Made available in DSpace on 2019-10-06T17:12:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-03 Alternative protein-coding transcripts of the RASSF1 gene have been associated with dual functions in human cancer: while RASSF1C isoform has oncogenic properties, RASSF1A is a tumour suppressor frequently silenced by hypermethylation. Recently, the antisense long non-coding RNA RASSF1 (ANRASSF1) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression mediated by PRC2 (Polycomb Repressive Complex 2). Here, we evaluated the methylation patterns of the promoter regions of RASSF1A and RASSF1C and the expression levels of these RASSF1 transcripts in breast cancer and breast cancer cell lines. As expected, RASSF1C remained unmethylated and RASSF1A was hypermethylated at high frequencies in 75 primary breast cancers, and also in a panel of three mammary epithelial cells (MEC) and 10 breast cancer cell lines (BCC). Although RASSF1C was expressed in all cell lines, only two of them expressed the transcript RASSF1A. ANRASSF1 expression levels were increased in six BCCs. In vitro induced demethylation with 5-Aza-2ʹ-deoxicytydine (5-Aza-dC) resulted in up-regulation of RASSF1A and an inverse correlation with ANRASSF1 relative abundance in BCCs. However, increased levels of both transcripts were observed in two MECs (184A1 and MCF10A) after treatment with 5-Aza-dC. Overall, these findings indicate that ANRASSF1 is differentially expressed in MECs and BCCs. The lncRNA ANRASSF1 provides new perspectives as a therapeutic target for locus-specific regulation of RASSF1A. Department of Genetics Institute of Biosciences São Paulo State University (Unesp) Department of Senology Amaral Carvalho Hospital Department of Biostatistics Institute of Biosciences São Paulo State University (Unesp) Department of Oncology Georgetown University Medical Center Faculdades Pequeno Préncipe e Instituto de Pesquisa Pelé Pequeno Príncipe Department of Clinical Genetics University Hospital Institute of Regional Health Research University of Southern Denmark Vejle Department of Genetics Institute of Biosciences São Paulo State University (Unesp) Department of Biostatistics Institute of Biosciences São Paulo State University (Unesp)

Data present here describe a comparative proteomic analysis among the malignant [primary breast tumor (PT) and axillary metastatic lymph nodes (LN)], and the non-tumor [contralateral (NCT) and adjacent (ANT)] breast tissues. Protein identification and quantification were performed through label-free mass spectrometry using a nano-liquid chromatography coupled to an electrospray ionization–mass spectrometry (nLC-ESI-MS/MS). The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier PXD012431. A total of 462 differentially expressed proteins was identified among these tissues and was analyzed in six groups' comparisons (named NCTxANT, PTxNCT, PTxANT, LNxNCT, LNxANT and PTxLN). Proteins at 1.5 log2 fold change were submitted to the Ingenuity® Pathway Analysis (IPA) software version 2.3 (QIAGEN Inc.) to identify biological pathways, disease and function annotation, and interaction networks related to cancer biology. The detailed data present here provides information about the proteome alterations and their role on breast tumorigenesis. This information can lead to novel biological insights on cancer research. For further interpretation of these data, please see our research article ‘Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer’ [2]. Keywords: Breast cancer, Contralateral non-tumor breast tissue, LC-ESI-MS/MS, Bioinformatics

In breast cancer, lymph node (LN) metastasis is one of the strongest prognostic factors at diagnosis. Therefore the identification of molecular markers with metastatic potential that promote the development of LN metastasis is of critical clinical relevance. In this study, we evaluated the copy number status of the FOSL1 , GSTP1 , NTSR1 , FADD and CCND1 genes by TaqMan assays in 137 breast cancer patients, 84 with LN metastasis (LN+) and 53 with no LN metastasis (LN−). The copy number data for four of these genes ( FOSL1 , GSTP1 , FADD and CCND1 ) were integrated with their mRNA expression levels in 31 patients. In both groups of patients, gains were the most frequent copy number alteration (CNA) observed, involving mainly the CCND1 , NTSR1 and FADD genes; mRNA overexpression was more commonly observed for the CCND1 and FADD genes. For the FADD gene in the LN+ group, gene expression was shown to be dependent on CNAs; for the other genes no association was found. In conclusion, increase copy number and mRNA overexpression of FOSL1 , GSTP1 , FADD , NTSR1 and CCND1 genes are frequently observed in primary breast tumors, and except for the FADD gene, they occur independently and irrespectively of the patients' LN axillary metastatic status.

Protein diversity arising from alternative mRNA splicing or post-translational modifications (PTM) such as phosphorylation, methylation etc. plays a vital role in the modulation of cellular functions. Mitotic kinases such as polo-like kinase 1 (PLK1) and aurora B (AURKB) are known to phosphorylate survivin, an inhibitor of apoptosis family member, to promote cell survival and proliferation. This triad is significantly overexpressed in triple negative breast cancer (TNBC), an aggressive breast cancer (BC) subtype, associated with high proliferative capacity, propensity for distant metastasis and chemotherapeutic resistance. TNBC disproportionately afflicts African-American (AA) women relative to European-American (EA) women. AAs demonstrate a much more aggressive disease course compared to EAs. Thus far, efforts to examine molecular underpinnings of racial disparity in TNBC have largely focused on differences in gene expression and epigenetic aspects. Herein, we demonstrate the role of survivin phosphorylation, an important PTM, in enhancing cell proliferation and reducing apoptosis in AA compared to EA TNBC cells. Using the TCGA breast cancer dataset, our in silico analysis of various mitotic kinases yielded a significantly higher gene expression of PLK1 (p=0.061) and AURKB (p=0.003) in AA (n=41) compared to EA TNBC (n=86) patients. Surprisingly, we found no race-related differences in survivin gene expression in this dataset (n=127, p=0.343). In addition, there was no statistical difference in protein levels of survivin in TNBC clinical samples (n=142, p=0.45) as measured immunohistochemically. These findings led us to rationalize that the differential activation of survivin in AA vs. EA TNBC cells is based on PTM, and not due to its differential gene/protein expression levels. We hypothesize that differential phosphorylation of survivin by mitotic kinases underlies enhanced proliferative and anti-apoptotic activity in AA relative to EA TNBC. Survivin phosphorylation at Ser-20 and Thr-117 is known to regulate the activity of the chromosome passenger complex facilitating mitosis and cell proliferation. Moreover, p-survivin Thr-117 inhibits X-linked inhibitor of apoptosis-Smac interaction, which in turn prevents caspase-9 activation, leading to anti-apoptosis. We observed an upregulation of phosphorylated substrates of PLK1 and AURKB viz. p-survivin Ser-20 & p-survivin Thr-117, respectively, in AA compared to EA TNBC cells. Survivin ablation significantly attentuated cell proliferation, cell cycle progression, and increased cell apoptosis in AA compared to EA TNBC cells. These data strongly support our premise that higher survivin phosphorylation confers enhanced proliferative capacity and anti-apoptosis in AA compared to EA TNBC cells, suggesting that modulating survivin PTM may be a viable therapeutic strategy for AA TNBC patients. Further studies intended to tease out the role of survivin phosphorylation in TNBC racial disparity are currently underway in our lab. Citation Format: Shriya Joshi, Chakravarthy Garlapati, Luciane Cavalli, Uma Krishnamurti, Shobhna Kapoor, Ritu Aneja. Mitotic kinases differentially phosphorylate survivin in African American TNBC: Clues for racial disparity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B070.

Women of African descent in the United States are twice as likely to develop triple-negative breast cancer (TNBC), compared to women of European descent. African American (AA) women with TNBC present a much more aggressive disease course relative to their European American (EA) counterparts. Thus, there is an urgent need to evaluate race-specific biomarkers and their molecular regulation to improve survival outcome in African American (AA)patients. Kinesin family member C1 (KIFC1), a minus-end-directed microtubule motor protein is involved in centrosomal clustering and chromosomal instability. KIFC1 is highly overexpressed (fold change=1.50, p=0.000392) in TNBC relative to non-TNBC. We previously showed that ablation of KIFC1 impaired proliferation and migration of AA TNBC cells to a greater extent than European American (EA) equivalent. Our published data also showed that the weighted index (WI) of nuclear KIFC1 (nKIFC1) is higher in AA TNBC (WI: 154.66 vs. 133.74, AA and EA, respectively, p = 0.036) and nKIFC1 is an independent poor prognosis biomarker exclusively for AA TNBC patients. While the centrosome clustering role of KIFC1 during mitosis is well-established, its predominant nuclear localization in interphase may underlie its previously unrecognized race-specific association. We and others have shown that nKIFC1 immunoprecipitates with various histones, helicases, and actinins. Immunoprecipitation (IP) of KIFC1 from nuclear fractions of TNBC cell lines followed by mass-spectrometry analysis revealed myosin heavy chain 9 (MYH9), a tumor suppressor protein that stabilizes p53 and promotes its nuclear retention, as the top nKIFC1 binding partner in AA TNBC cell lines (MDA-MB-468 and HCC1806). However, we observed a rather low affinity (rank 5th) or no binding of MYH9 with nKIFC1 in EA TNBC cell lines (MDA-MB-436, HCC1937). Our IP-MS data suggest that a) the relative abundance of MYH9 in nKIFC1 immunoprecipitates is higher in the AA TNBC cell lines and b) MYH9 expression is higher in AA TNBC compared to EA TNBC cells. We hypothesize that nKIFC1 impairs the tumor suppressor function of MYH9 more effectively in AA compared to EA TNBC cells. Intriguingly, KIFC1 immunoprecipitates also showed the presence of beta, alpha-, and gamma actin along with several dead box helicases such as DDX5, DDX17, exclusively in AA TNBC cell lines, suggesting that a combination therapy regimen of KIFC1 inhibitor (SR31527, CW069, and AZ82) and taxane/anthracycline/platinating agent might be more effective in AA than in EA TNBC cells. RNA seq analyses to identify other KIFC1partners using KIFC1 knock out using CRISPR-CAS9 approach in AA and EA TNBC cell lines are currently underway. Citation Format: Chakravarthy Garlapati, Shriya Joshi, Luciane Cavalli, Uma Krishnamurti, Shobhna Kapoor, Ritu Aneja. Myosin heavy chain 9 (MYH9) is a novel interacting partner of kinesin family member C1 (KIFC1) in African American TNBC: Implications for racial disparity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A070.

Triple-negative breast cancer (TNBC)—defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative—is a highly aggressive form of breast cancer prevalent in African-American (AA) women. Current treatment strategies rely on cytotoxic chemotherapy because ER- and HER2-targeted therapies are ineffective in TNBC, leaving a tremendous need for new effective therapies with less toxicity. Nuclear receptors are highly druggable targets. Classical examples include ligand-regulated receptors like ER, but there is opportunity for orphan nuclear receptors (ONR) to be studied as potential targets for cancer therapy. Our lab has published that increased mRNA expression of ONR estrogen-related receptor beta (ERRβ, gene symbol ESRRB) correlates with better recurrence- and distant metastasis-free survival in women with TNBC/basal-like breast cancer. We also showed that treatment with a small-molecule agonist ligand for ERRβ (DY131) has growth-inhibitory and antimitotic activity in TNBC cell lines. The goal of our current work is to comprehensively characterize ERRβ copy number, mRNA, protein expression, and ERRβ function in TNBC. ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by Agilent SurePrint G3 Human CGH Microarray in a cohort AA and Caucasian (CA) women. ESRRB mRNA expression was determined in The Cancer Genome Atlas (TCGA) Breast Cancer RNAseq data. Association of ESRRB mRNA with overall survival was determined in Illumina gene expression array data from METABRIC. ERR protein expression is being assessed by immunohistochemistry (IHC) in a tissue microarray of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+). Ongoing studies are focusing on genetic modification of cell lines, specifically the overexpression of ERRβ in nontransformed mammary epithelial and TNBC cell lines. DNA: In both TNBC and nonTNBC, AA patients had a markedly higher frequency of ESRRB copy number loss than CA patients (2 *p=0.012 for TNBC, p=0.052 for nonTNBC). RNA: In TCGA breast cancer patients by PAM50 subtype, TNBC/basal-like patients have significantly lower ESRRB mRNA expression than Luminal A patients (*p=0.0015). Among systemically untreated patients in the METABRIC dataset, low ESRRB mRNA is significantly associated with poor overall survival in TNBC (hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Protein: Analysis of ERR protein expression by IHC is ongoing. Function: Focusing on overexpression of ERRβ in cell lines, we aim to analyze multiple phenotypes including cell proliferation, cell invasion and migration, and differential gene expression. Conclusions: Breast tumors from AA women are significantly more likely to have reduced ESRRB copy number vs. CA women. TNBC patients have lower ESRRB mRNA expression and low ESRRB mRNA expression predicts for poor overall survival in TNBC. These data suggest that ERRβ expression has prognostic value in breast cancer, particularly in TNBC. Citation Format: Aileen I. Fernandez, Garrett Graham, Bálazs Györffy, Luciane R. Cavalli, Rebecca B. Riggins. ERRβ: Validating a novel target for triple-negative breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C103.

Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (n = 12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [n = 396 (PTxNCT) and n = 410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. Significance The proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.

Breast cancer is the most common and the leading cause of female mortality among South African (SA) women. Several non‑biological and biological risk factors may be attributed to their observed high mortality rate; however, the molecular profiles associated with their breast tumors are poorly characterized. The present study examined the patterns of genome-wide copy number alterations (CNAs) and their potential impact on functional cellular pathways targeted by cancer driver genes in patients with breast cancer from the Western Cape region of SA. Array-comparative genomic hybridization analysis, performed in 28 cases of invasive breast cancer, revealed a mean number of 8.68±6.18 CNAs per case, affecting primarily the Xp22.3 and 6p21-p25 cytobands (57.14% of the cases), followed by 19p13.3-p13.11 (35.7%), 2p25.3-p24.3, 4p16.3-p15.3, 8q11.1-q24.3 and 16 p13.3-p11.2 (32.14%). Functional enrichment analysis of genes and microRNA targets mapped in these affected cytobands revealed critical cancer-associated pathways, including fatty acid biosynthesis and metabolism, extracellular matrix-receptor interaction, hippo and tumor protein p53 signaling pathways, which are regulated by known cancer genes, including CCND1, CDKN1A, MAPK1, MDM2, TP53 and SMAD2. An inverse correlation was observed among the number of CNAs and tumor size and grade; CNAs on the 4p and 6p cytobands were also inversely correlated with tumor grade. No association was observed in the number of CNAs and/or the affected cytobands and the different ethnic groups of the SA patients, indicating that their tumor genome is affected by CNAs, irrespectively of their genetic descent. Additional genomic tumor profiling in SA and other Sub-Saharan African patients with breast cancer is required to determine the associations of the CNAs observed with prognosis and clinical outcome.

Breast cancer is one of the main causes of cancer death among South African women. Although several risk factors can be attributed to the observed high mortality rate, the biology of the tumors is not extensively investigated. Copy number gain of the DLX4 homeobox gene has been observed in breast cancer in association with poor prognosis and specific racial groups. Therefore, we aimed to assess the copy number and prognostic role of DLX4 in breast cancer from South African patients. Due to the co-location of ERBB2 and DLX4 in the 17q21 region, its copy number was also evaluated. Our results in the analysis of 66 cases demonstrated copy number gains of DLX4 and ERBB2 in 24.1 and 29.7% of the cases, respectively. Linear regression analysis showed no dependency between the copy number alterations in these genes. Although not significant, patients with DLX4 and ERBB2 gains presented a higher frequency of advanced-grade tumors. In addition, copy number alterations of these genes were not significantly differently observed in the 3 main racial groups of the Western Cape population: Colored, White, and Black. These findings indicate that gains of DLX4 and ERBB2 occur in South African breast cancer patients irrespectively of their race and factors known to influence prognosis.

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

The key molecular events required for the formation of ductal carcinoma in situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here, we show that the nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal three-dimensional mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both before DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ breast cancer-initiating cell (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. The loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, human epidermal growth factor receptor 2 (HER2) and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 has a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through the maintenance of BCIC, facilitates formation of DCIS, a necessary step before development of invasive disease.

We examined a girl presenting neuropsychomotor developmental delay and multiple malformations including antenatal and postnatal growth retardation, congenital heart defect, and facial dysmorphisms. Cytogenetic analysis was performed on peripheral blood lymphocytes with the GTG-banding technique, which revealed an unbalanced translocation: 46,XX,der(13)(13pter→13q34::3p24→3pter)pat. Karyotype analysis of the father demonstrated a balanced translocation, 46,XY,t(3;13)(p24;q34), indicating the inheritance of the derivative chromosome 13. The mother karyotype was normal. We suggest that most of the structural malformations seen in this patient are due to the 3p trisomy, while the neuropsychomotor alterations are a consequence of both chromosome aberrations.

The 2 main histologic types of infiltrating breast cancer, invasive lobular and invasive ductal carcinoma, are morphologically and clinically distinct. Studies revealed that different patterns of gene expression and loss of heterozygosity can also distinguish these 2 subtypes. A whole-genome study using single nucleotide polymorphism array found a significantly higher frequency of loss of heterozygosity on 3p in invasive ductal carcinoma when compared with invasive lobular carcinoma. In this study, we performed a loss of heterozygosity analysis of the 3p chromosome region in ductal and lobular breast tumors. Seven microsatellite markers were evaluated in a series of 136 sporadic breast cancer cases (118 invasive ductal carcinoma and 18 invasive lobular carcinoma) and correlated with clinical-histopathologic parameters from the patients. A significantly higher frequency of loss of heterozygosity was observed in invasive ductal carcinoma (65.3%) when compared with invasive lobular carcinoma (38.9%). When the markers were analyzed separately, loss of heterozygosity at 3 of them, D3S1307 in 3p26.3, D3S1286 in 3p24.3, and D3S1300 in 3p14.2, were significantly more frequent in ductal than in lobular tumors. D3S1307 marker showed the highest frequency of loss of heterozygosity in invasive ductal carcinoma (46.2%), and associations between loss of this marker and loss of estrogen and progesterone receptors were found in these samples. Our results confirm the observations that invasive ductal carcinoma has a higher frequency of loss of heterozygosity events across the 3p region than invasive lobular carcinoma and show that specific losses on 3p26.3, 3p24.3, and 3p14.2 regions are more frequent in ductal than in lobular tumors. We discuss our data in relation to the known tumor suppressor genes that are mapped at the 3p loci investigated and their present relevant roles in breast cancer.