TITLE of PART I: MICRORNA-205 IS A TUMOR SUPPRESSOR IN BREAST CANCER Major Professor: Dr. Yinyuan Mo MicroRNAs as a class of novel negative regulators have drawn intensive attention since their existence in mammals was discovered in 2001. As endogenous small non-coding RNAs, miRNAs are capable of silencing gene expression at the post-transcriptional level by translation blocking or/and mRNA degradation. MicroRNA has been reported to involve in diverse biological events. Although miRNA deregulation has been observed in many types of cancers including breast cancer, only few of these de-regulated miRNAs have been confirmed to be related with breast cancer progression. This study is to investigate the association of miRNAs with the development of breast cancer. In this study, we report that mir-205 is significantly under-expressed in breast tumors compared to the matched normal breast tissues. Similarly, breast cancer cell lines including MCF-7 and MDA-MB-231 express a much lower level of mir-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of mir-205 significantly inhibits cell proliferation and anchorage-independent growth as well as cell invasion. Furthermore, mir-205 was shown to suppress lung metastasis in a mouse model. Finally, western blot combined with the luciferase reporter assays demonstrated that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets of mir-205 and such mir-205-mediated suppression is likely through the direct interaction with the putative mir-205 binding site in the 3'-untranslated region (3-UTR) of ErbB3 and VEGF-A, suggesting that down-regulation of ErbB3 and VEGFA likely contributes to the suppression role of mir-205 in breast cancer cells. Together, these results suggest that mir-205 is a tumor suppressor in breast cancer. Given that mir-205 is downregulated in breast tumor specimens and breast cancer cell lines, it would be interesting to determine how mir-205 is regulated in cancer cells. Supprisingly, we found that agents which have been previously shown to induce other miRNAs, such as p53 induction, the phenolic compound kaempferol, hypoxia, and ROS stress have little effect on mir-205 expression, whereas de-methylation and UV treatment only a moderate effect on mir-205. Therefore, our studies indicate that mir-205 is a tumor suppressor in breast cancer progression, however, regulation of mir-205 still remains to be elucidated. Knowledge gained from these studies will provide more comprehensive understanding of how miRNAs are associated with tumor initiation and progression in breast cancer and how miRNAs are de-regulated during cancer development, and as a result, mir-205 may serve as a novel target for cancer therapy. TITLE of PART II: REGULATION OF ESTROGEN-INDEPENDENT GROWTH BY MICRORNAS Major Professor: Dr. Yinyuan Mo Tamoxifen (TAM) has proven to be effective in the treatment of breast cancer. The primary target of TAM in vivo is estrogen receptor (ER), and thus levels of ER expression are the best predictors of benefit from TAM. Unfortunately, over 30% of ER-positive tumors fail to respond to TAM therapy; moreover, those breast tumors that initially respond to TAM will frequently develop resistance to the treatment. Although it is believed that this is likely due to the activation of estrogen-independent signaling, the precise molecular mechanism is not fully understood. This poses a significant challenge to the determination of treatment options for ER positive breast tumors. In this study, we performed in vivo selection experiments using the breast cancer ER positive MCF-7 cells infected with a pooled human microRNA library. It is well known that MCF-7 cells do not grow tumors in a xenograft mouse model unless estrogen is provided. We found that the MCF-7 cells infected with the microRNA library, but not the vector control, did grow tumors, suggesting that certain microRNAs are able to support the estrogen-independent growth. To characterize these microRNA-induced tumors, we recovered cells from the tumors in cell culture and found that they are more resistant to TAM than the MCF-7 cells with vector control. Moreover, they are able to grow in estrogen-free medium. Further analysis revealed that these recovered cells are ER positive with highly activated pAkt. By genomic DNA based real-time PCR, we identified that 6 miRNAs have been enriched in the tumor genome. Co-infection of these 6 enriched miRNAs is able to confer MCF-7 cells estrogen-independent growth and tamoxifen resistance. Further analysis indicated that mir-101 alone is sufficient to induce in vitro estrogen-independent growth and tamoxifen resistance. Together, these results suggest that microRNAs are important players in regulating the estrogen-independent growth and thus, identifying such microRNAs and understanding the underlying mechanisms will provide new insight into estrogen-independent tumor growth and TAM resistance, and thus, these microRNAs may serve as novel targets for breast cancer therapy.

Thesis (M.D.)—Boston University

Over the past two decades, the mammalian Ste20-like kinase (SLK) has been characterized for its role in regulating cellular migration, proliferation and apoptosis in fibroblasts and myoblasts. In mammary epithelial cells, SLK has been shown to be required for efficient epithelial-to-mesenchymal transition and to be activated downstream of the HER2/Neu-oncogene to control chemotactic cellular migration. Here, we assessed the role of SLK in HER2/Neu-induced mammary tumorigenesis in vivo. As SLK is activated downstream of HER2/Neu, we hypothesized that the loss of SLK would significantly delay tumor progression in a mouse model of HER2-positive breast cancer. As we have shown that global attenuation of SLK kinase activity results in embryonic lethality, a conditional SLK knockout mouse model was generated. To study the role of SLK in HER2-positive breast cancer, we crossed these conditional SLK knockout mice with mice expressing HER2/Neu linked to Cre recombinase in the mammary luminal epithelium. Unexpectedly, we have demonstrated that conditional deletion of SLK significantly accelerates Neu-induced mammary tumor onset and decreases overall survival. SLK deletion results in the induction of Sox10 which drives mammary stem/progenitor activity and cooperates with HER2/Neu to drive tumor growth. Using the Cancer Genome Atlas, we have supported previous findings and validated Sox10 as a potential biomarker of the Triple-negative Breast Cancer subtype. Furthermore, we have uncovered that SLK deletion results in enhanced activation of both PDK1 and AKT. We provide evidence that Sox10 induction requires signaling through a novel AKT/Sox9-dependent pathway following SLK deletion. Taken together, our data suggests that SLK may be required to maintain cells in a fully differentiated state and that loss of SLK in HER2/Neu-induced breast cancer drives a more basal/stem-like phenotype through the induction of Sox10.

Made available in DSpace on 2014-06-11T19:32:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T19:43:36Z : No. of bitstreams: 1 arantesjr_jc_dr_botfm.pdf: 447982 bytes, checksum: f2d80a448fdaad2a52ea33a40154ad2e (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / O câncer de mama acomete muitas mulheres em todo o mundo e as metástases são a principal causa de morte. Conhecimentos acerca do comportamento biológico são úteis para aprimorar a abordagem, principalmente terapêutica. Os perfis histológico e imuno-histoquímico podem nos permitir melhor compreensão dos fenômenos que determinam a evolução da doença. Objetivo: Comparar lesões palpáveis e não palpáveis para determinar agressividade biológica, baseado nos parâmetros histopatológicos e imuno-histoquímicos. Metodologia: Foram estudados 296 (duzentos e noventa e seis) casos de câncer de mama, confirmados pela histopatologia, separados em dois grupos: Grupo 1: Lesões não palpáveis, detectadas exclusivamente por mamografia (microcalcificações, nódulo ou distorção arquitetural) ou por achado incidental e Grupo 2: Lesões palpáveis (nódulo, massa ou condensação), em estudo retrospectivo, analítico e horizontal. Foram avaliados critérios histopatológicos (Grau Nuclear, Formação Tubular e Índice Mitótico) e imuno-histoquímicos (Receptores de Estrogênio, Receptores de Progesterona, Ki67, p53 e c-erbB-2) de agressividade, comparando-se os dois grupos, com tratamento estatístico (índice de significância p<0,05). Resultados: Avaliados os dois grupos indistintamente, observou-se que a maior expressão dos Receptores Hormonais (RE e RP) esteve relacionada com Grau Nuclear, Formação Tubular e Índice Mitótico baixos. Super-expressão de Ki67 foi prevalente nos graus histopatológicos mais elevados. c-erbB-2 e p53, embora não estatisticamente significativo, apresentaram a mesma tendência do Ki67 em relação às variáveis histopatológicas. O Grupo 1 apresentou menor incidência de Grau Nuclear, Formação Tubular e Índice Mitótico elevados que o Grupo 2. O Grupo 1 apresentou maior expressão dos Receptores Hormonais (p<0,05), enquanto o Grupo 2 apresentou maior expressão de Ki67(p<0,05) / The breast cancer commits many women throughout the world, and the metastases are the main cause of deaths. The knowledge about the biologic aggressiveness is useful to improve the treatment and the histopathological and immunohistochemical profiles can help to understand the events that determine the outcome. Objective: To compare two groups of patients with breast cancer (Group 1: non-palpable lesions; Group 2: palpable lesions), based in histopathological and immunohistochemical parameters to determine the biologic aggressiveness. Methods: We have studied 296 (two hundred ninety six) cases of breast cancer, diagnosed by histopathology, divided in two groups: Group 1- non-palpable lesions, exclusively detected by mammography (microcalcifications, nodule or architectural distortion) or with incidental finding and Group 2- palpable lesions (Nodule or mass), in retrospective, analytical and horizontal analysis. We evaluated histopathological parameters (nuclear grade, tubularity and mitotic counts) and immunohistochemical markers(Estrogen Receptors, Progesterone Receptors, p53, Ki67 and c-erbB-2), comparing the two groups, with statistic analysis (p<0.05). Results: The bigger the expression of estrogen and progesterone receptors the smaller the nuclear grade, tubularity and mitotic counts. On the contrary, the bigger the expression of Ki67 the higher the nuclear grade, tubularity and mitotic counts. We observed that c-erbB-2 and p53 expression showed the same tendency of the Ki67 in relation to the histopathologic parameters. Group 1 showed less incidence of elevated nuclear grade, tubularity and mitotic counts, bigger expression of Estrogen and Progesterone Receptors(p<0.05). Group 2 showed higher expression of Ki67 (p<0.05). The c-erbB-2 expression was a little bit higher in Group 2 than in Group 1(p=0.055). The p53 expression didnot show differences between the groups.

Orientador: Gilberto Uemura / Banca: José Ricardo Paciência Rodrigues / Banca: Helena Cristina da Silva / Resumo: O câncer de mama é um problema de saúde pública, com 49.470 casos em 2005 no Brasil. A mamografia é o método de escolha para rastreamento e diagnóstico precoce do câncer de mama. O sistema BI RADS veio tentar organizar e padronizar os laudos mamográficos. O VPP do Bl-RADS IV varia de 2 a 90% e da categoria V > 90%. Objetivos: avaliar as principais características das lesões mamográficas suspeitas e calcular o VPP da categoria IV e V. Sujeitos e métodos: foram estudados 309 laudos mamográficos de Bl-RADS IV e V e correlacionados com seus resultados de histopatológicos. A análise estatística foi o teste diagnóstico do VPP. Resultados: idade média 54 anos, mama esquerda acometida em 165 (53,4%) pacientes e mama direita em 144 (46,6%) pacientes, BI RADS IV 265 (85,8%), Bl-RADS V 44 (14,2%), lesões benignas 163 (52,8%), lesões proliferativas de risco 48 (15,5%), lesões malignas 98 (31,7%). Os principais achados mamográficos na categoria IV foram as microcalcificações 163 (61,5%) e na categoria V foram os nódulos em 22 (50,0%). A modalidade de biopsia mais usada na categoria IV foi o agulhamento mamário em 188 (70,9%) e na categoria V foi à biopsia per cutânea em 16 (36,4%). O carcinoma ductal infiltrante foi o tipo histológico mais freqüente na categoria IV em 45 (78,9%) e na categoria V em 35 (85,4%) pacientes. O VPP para a categoria IV foi de 39,2% e da categoria V de 95,5%. Conclusão: Concluímos que os principais achados mamográficos na categoria IV foram as microcalcificações, com VPP desta categoria de 39,2%; na categoria V foram os nódulos os principais achados mamográficos, com VPP de 95,5%, valor este que confirma esta categoria como alta probabilidade de malignidade. / Abstract: Breast cancer is a serious world health problem. The mammography is the better method for the screening of earlier breast cancer. The Bl-RADS system was introduced to standardize the report results. Bl-RADS IV has a Positive Predictive Value (PPV) ranged from 2 to 90% and over 90% to category V. Objectives: to evaluate the principal characteristics of the suspicious mammographic lesions and calculate PPV of the Bl-RADS category IV and V. Subjects and methods: it was evaluated 309 reports of mammographic abnormalities classified as Bl-RADS IV and V; and their correlations with biopsies results. Resulted: medium age was 54 years, the lesions occurred in 165 left-sided breast (53.4%) and in 144 right-sided breast (46.6%). Bl-RADS IV pattern was identified in 265 (85.8%) reports and BI-RADS V in 44 (14.2%). Benign lesions were found in 163 reports (52.8%), proliferative risk lesions were found in 48 (15.5%), malign lesions were found in 98 (31.7%). The principal mammographic lesion was the microcalcification in category IV and nodules were in category V. The most used procedure for diagnosis was core biopsy in 204 patients (66%). The invasive ductal carcinoma was the predominant histological type in malignant breast tumors. PPV of 39.2% was observed for BI-RADS IV and for BI-RÃDS V was 95.5%. Conclusion: the principal mammographic pattern lesion in the Bl-RADS IV was the microcalcification, with PPV of 39.2% and the nodules were the principal mammographic pattern lesions in the BI-RADS V with PPV of 95.5%. It confirms the high probability of malignancy in BI-RADS V category. / Mestre

Resumo: O câncer de mama acomete muitas mulheres em todo o mundo e as metástases são a principal causa de morte. Conhecimentos acerca do comportamento biológico são úteis para aprimorar a abordagem, principalmente terapêutica. Os perfis histológico e imuno-histoquímico podem nos permitir melhor compreensão dos fenômenos que determinam a evolução da doença. Objetivo: Comparar lesões palpáveis e não palpáveis para determinar agressividade biológica, baseado nos parâmetros histopatológicos e imuno-histoquímicos. Metodologia: Foram estudados 296 (duzentos e noventa e seis) casos de câncer de mama, confirmados pela histopatologia, separados em dois grupos: Grupo 1: Lesões não palpáveis, detectadas exclusivamente por mamografia (microcalcificações, nódulo ou distorção arquitetural) ou por achado incidental e Grupo 2: Lesões palpáveis (nódulo, massa ou condensação), em estudo retrospectivo, analítico e horizontal. Foram avaliados critérios histopatológicos (Grau Nuclear, Formação Tubular e Índice Mitótico) e imuno-histoquímicos (Receptores de Estrogênio, Receptores de Progesterona, Ki67, p53 e c-erbB-2) de agressividade, comparando-se os dois grupos, com tratamento estatístico (índice de significância p<0,05). Resultados: Avaliados os dois grupos indistintamente, observou-se que a maior expressão dos Receptores Hormonais (RE e RP) esteve relacionada com Grau Nuclear, Formação Tubular e Índice Mitótico baixos. Super-expressão de Ki67 foi prevalente nos graus histopatológicos mais elevados. c-erbB-2 e p53, embora não estatisticamente significativo, apresentaram a mesma tendência do Ki67 em relação às variáveis histopatológicas. O Grupo 1 apresentou menor incidência de Grau Nuclear, Formação Tubular e Índice Mitótico elevados que o Grupo 2. O Grupo 1 apresentou maior expressão dos Receptores Hormonais (p<0,05), enquanto o Grupo 2 apresentou maior expressão de Ki67(p<0,05) / Abstract: The breast cancer commits many women throughout the world, and the metastases are the main cause of deaths. The knowledge about the biologic aggressiveness is useful to improve the treatment and the histopathological and immunohistochemical profiles can help to understand the events that determine the outcome. Objective: To compare two groups of patients with breast cancer (Group 1: non-palpable lesions; Group 2: palpable lesions), based in histopathological and immunohistochemical parameters to determine the biologic aggressiveness. Methods: We have studied 296 (two hundred ninety six) cases of breast cancer, diagnosed by histopathology, divided in two groups: Group 1- non-palpable lesions, exclusively detected by mammography (microcalcifications, nodule or architectural distortion) or with incidental finding and Group 2- palpable lesions (Nodule or mass), in retrospective, analytical and horizontal analysis. We evaluated histopathological parameters (nuclear grade, tubularity and mitotic counts) and immunohistochemical markers(Estrogen Receptors, Progesterone Receptors, p53, Ki67 and c-erbB-2), comparing the two groups, with statistic analysis (p<0.05). Results: The bigger the expression of estrogen and progesterone receptors the smaller the nuclear grade, tubularity and mitotic counts. On the contrary, the bigger the expression of Ki67 the higher the nuclear grade, tubularity and mitotic counts. We observed that c-erbB-2 and p53 expression showed the same tendency of the Ki67 in relation to the histopathologic parameters. Group 1 showed less incidence of elevated nuclear grade, tubularity and mitotic counts, bigger expression of Estrogen and Progesterone Receptors(p<0.05). Group 2 showed higher expression of Ki67 (p<0.05). The c-erbB-2 expression was a little bit higher in Group 2 than in Group 1(p=0.055). The p53 expression didnot show differences between the groups. / Orientador: Gilberto Uemura / Coorientador: Rogério Estevam Farias / Doutor

Objective: In this study, I have directly tracked the progression of angiogenesis for three different types of breast cancer cell lines; MDA-MB-231, MCF-7, and MDA-MB-468. Each of these cell lines is known to overexpress different receptors, which may affect a tumor’s growth rate and perhaps its ability to undergo angiogenesis. Here, I measure and compare the growth, extent, and time of onset for angiogenesis. Methods: I used SCID mice to profile each of the different breast cancer cell lines. The growth rate of each tumor, along with its blood vessel development, was monitored and imaged using lipid-coated microbubbles and contrast-enhanced ultrasound (CEUS). A Vevo 2100 pre-clinical ultrasound machine was used for the imaging experiments. To track development of angiogenesis, mice were injected with perfluorobutane gas microbubbles of 1-2 microns diameter. Bubble perfusion into the tumor is an indicator of the presence of blood vessel formation. A custom image analysis program was developed in Matlab™ to eliminate breathing artifacts and track microbubble motion based on their high temporal frequency signature ("flicker"). Results: My experiments demonstrated that, although different cell lines grow at different rates, microbubbles begin to penetrate the tumor when it reaches approximately a size of approximately 3 mm in diameter. Therefore, the onset of angiogenesis occurred at different times (MCF-7 occurring first at around 9 days, MDA-MB-468 occuring at 12 days post inoculation, and MDA-MB-231 occurring at 17 days post tumor cell inoculation). Matlab™ analysis demonstrates consistent angiogenic behavior among the three cell lines. Conclusion: For all cell lines, angiogenesis started when the volume of the tumor was approximately 21.76 mm³, consistent with previous studies. As angiogenesis progressed, there was a drop in tumor blood flow. This can be explained by the sudden influx of oxygen when angiogenesis first begins. This momentarily inhibits new blood vessel formation while the tumor continues to steadily grow. After this sudden drop, tumor vascularization resumes a steady increase.

Mexican American women are at particular risk of being diagnosed with breast cancer at a later stage. Risk factors include genetics, limited screening practices, and delayed treatment. Evidence has shown culture to be an important factor influencing screening beliefs, health care behaviors, and breast cancer knowledge. A culturally and linguistically appropriate educational video about breast cancer and breast cancer screening recommendations was created, in both the English and Spanish languages, to engage Mexican American women and explore their perceptions and feedback about the culturally tailored intervention. Qualitative descriptive methodology was used to explore Mexican American women’s perceptions of a culturally and linguistically appropriate educational video about breast cancer and the importance of breast cancer screenings. Using snowball recruitment from a Spanish language breast cancer support group, eight Mexican origin women participated in a focus group interview. The interview was conducted in the Spanish language. The video production integrated Mexican cultural values and used them as instruments to present information about breast cancer and breast cancer screening recommendations. An extensive literature review and a theoretical underpinning helped guide the project purpose and intervention. The findings supported the importance of production of a culturally and linguistically appropriate education video to educate Mexican American women about breast cancer and the importance of breast cancer screenings. Analysis of the focus group discussion identified an overarching theme of “Language, Identity, Values” which supports the underlying premise that the information needed to be presented in the language spoken by the population of focus, in ways they could identify with, using cultural values to under pin the messages presented in the video. The implications for nurse practitioners, centers on the importance of employing Mexican cultural values when imparting knowledge. More studies like this one can help further identify the impact of Mexican cultural values on health care.

Magister Scientiae - MSc / Breast cancer (BC) is the most common cancer in women worldwide, and is the second most common cancer in the world, responsible for more than 500 000 deaths annually. Estimates are that 1 in 8 women will develop BC in their lifetime. In South Africa, BC in women affects about 16.6 % of the population and could see a 78 % increase in cases by 2030. The failure of conventional diagnostic tools to detect BC from an early onset has revealed the need for diagnostic tools that would enable early diagnosis of BC. The current diagnostic tools include breast self-examination, mammography magnetic resonance imaging, ultrasonography and serum biomarkers; BRACA1, BRACA2, HER2. These conventional methods lack sensitivity, specificity and positive predictive value, and some of these diagnostic tools may be expensive and quite invasive. Therefore, novel diagnostic tools such as microRNAs which address the short comings of current methods are required for early diagnosis as well as BC management. MicroRNAs are a class of non-coding RNA molecules, which are important in RNA stability and gene expression. Various methodologies have been employed to identify novel microRNAs for diagnostics such as bioinformatics, also referred to as in silico analysis. The aim of this study is to identify novel microRNAs that can potentially detect BC at its earliest stage.

This thesis assesses the main risk factors contributing to contralateral breast cancer(CBC) amongst high-risk breast cancer patients with a view to formulating clinicallyuseful guidelines. The work has focused on several keys areas; a literature review ofthe various factors contributing to CBC, changing trends towards increasingnumbers of contralateral risk-reducing mastectomies (CRRMs), internationalvariations amongst breast surgeons’ attitudes towards risk-reducing mastectomy(RRM), attitudes towards CRRM amongst UK breast and plastic surgeons,assessment of CBC risk amongst BRCA1/2 mutation carriers and finally theformation of the ‘Manchester Guidelines for CRRM’.Breast cancer patients harbouring mutations in high penetrance genes (i.e. BRCA1/2,TP53, CHEK2, PALB) have the highest risk of developing breast cancer. A positivefamily history also increases the risk of subsequent breast cancer, with not muchevidence to support variation in risk with histological type. Risk reducing strategiesinclude anti-endocrine treatment, risk-reducing bilateral salpingo-oophorectomy(RRBSO) and CRRM with the former likely to account for the global trend ofdecreasing rates of CBC.Over the last decade, rates of CRRM have trebled in the USA – such a clear trendhas not yet been confirmed in Europe. Factors driving this trend include young ageat diagnosis, histological type (lobular carcinoma, lobular carcinoma in situ [LCIS]and ductal carcinoma in situ [DCIS]) and female surgeons. A direct comparison(USA v 4 European countries) found that American surgeons overall had a greaterknowledge of cancer genetics and nearly all (including Dutch and British surgeons)had positive attitudes towards RRM.A proportion of British surgeons were quoting inaccurate levels of CBC risk to theirpatients. Practices in the UK varied regarding CRRM – only 58% of surgeons alwaysdiscussed these cases in the MDT, with less than a third ever seeking apsychological or formal genetic assessment. Surgeons primarily offered thisprocedure to high-risk patients (gene mutation carriers or positive family history)but felt that the main reason patients requested CRRM was to alleviate anxiety. Studying over 1000 breast cancer patients who also had a mutation in either BRCA1or BRCA2 gene revealed that the risk of CBC was approximately 2-3% per year, forat least 2 decades. Young age at first breast cancer development (<40 years) affectedthis risk most. The effect bilateral risk-reducing salpingo-oophorectomy (BRRSO)was initially significant in an unadjusted analysis, but when accounting for delayedentry BRRSO did not appear to affect CBC risk. The use of SNPs was not able tostratify risk of CBC further. By considering the above, the Manchester Guidelines for CRRM have beenformulated. This 5-step protocol allows clinicians to objectively assess the risk ofCBC based on the evidence base and makes suggestions of a multi-disciplinaryapproach to managing requests for CRRM. Several breast units in the UK havealready adopted these guidelines and future studies hope to validate them so thatthey can be used more widely.