Protein Kinase D3 promotes the cell proliferation by activating the ERK1/c‐MYC axis in breast cancer

Breast cancer is the second leading death cause of cancer death for all women. Previous study suggested that Protein Kinase D3 (PRKD3) was involved in breast cancer progression. In addition, the protein level of PRKD3 in triple‐negative breast adenocarcinoma was higher than that in normal breast tissue. However, the oncogenic mechanisms of PRKD3 in breast cancer is not fully investigated. Multi‐omic data showed that ERK1/c‐MYC axis was identified as a major pivot in PRKD3‐mediated downstream pathways. Our study provided the evidence to support that the PRKD3/ERK1/c‐MYC pathway play an important role in breast cancer progression. We found that knocking out PRKD3 by performing CRISPR/Cas9 genome engineering technology suppressed phosphorylation of both ERK1 and c‐MYC but did not down‐regulate ERK1/2 expression or phosphorylation of ERK2. The inhibition of ERK1 and c‐MYC phosphorylation further led to the lower protein level of c‐MYC and then reduced the expression of the c‐MYC target genes in breast cancer cells. We also found that loss of PRKD3 reduced the rate of the cell proliferation in vitro and tumour growth in vivo, whereas ectopic (over)expression of PRKD3, ERK1 or c‐MYC in the PRKD3‐knockout breast cells reverse the suppression of the cell proliferation and tumour growth. Collectively, our data strongly suggested that PRKD3 likely promote the cell proliferation in the breast cancer cells by activating ERK1‐c‐MYC axis.     

The oestrogen paradox: a hypothesis

Epidemiological and observational studies suggest that oestrogens, when used as hormonal therapy in post-menopausal women, can increase the risk of breast cancer if used long term. However, more recent data suggest that short-term use in sub-groups of post-menopausal women significantly decreases the risk of breast cancer. This beneficial effect is also observed when high-dose oestrogen is administered to post-menopausal women with breast cancer to cause tumour regression, a phenomenon which commonly occurs. We consider these divergent responses to oestrogen to represent a "paradox". Data from our own and other investigative groups suggest a hypothesis to explain this paradox. Deprivation of oestradiol in model systems causes cells to adapt and to undergo apoptosis in response to oestrogen. This occurs through the Fas/Fas ligand death receptor pathway and through alterations in apoptotic mechanisms mediated by mitochondria. This process of programmed cell death may explain the regression of established breast cancer with oestrogen administration and the diminution in the rate of new breast cancer diagnoses recently reported. Our hypothesis is based upon pathological data indicating the presence of a "reservoir" of undiagnosed breast cancer in the population of women who would be starting on oestrogens as menopausal hormonal therapy. The long-term increased risk of breast cancer may then reflect different mechanisms. Oestrogens can cause mutations through enhancement of the rate of cell division and concomitantly the error rate in DNA replication. In addition, oestrogens can be metabolised to directly genotoxic compounds. These carcinogenic processes take much longer, since a number of mutations must accumulate before resulting in breast cancer. These hypotheses regarding oestrogen-induced apoptosis in the short term and carcinogenesis in the long term now require rigorous verification but would serve to explain the "oestrogen paradox".     

A New Mouse Model for the Study of Human Breast Cancer Metastasis

Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD scid gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.     

Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat

Compared with the general population, women who have inherited a germline mutation in the BRCA1 gene have a greatly increased risk of developing breast cancer. However, there is also substantial interindividual variability in the occurrence of breast cancer among BRCA1 mutation carriers. We hypothesize that other genes, particularly those involved in endocrine signaling, may modify the BRCA1-associated age-specific breast cancer risk. We studied the effect of the CAG repeat-length polymorphism found in exon 1 of the androgen-receptor (AR) gene (AR-CAG). AR alleles containing longer CAG repeat lengths are associated with a decreased ability to activate androgen-responsive genes. Using a sample of women who inherited germline BRCA1 mutations, we compared AR-CAG repeat length in 165 women with and 139 women without breast cancer. We found that women were at significantly increased risk of breast cancer if they carried at least one AR allele with >/=28 CAG repeats. Women who carried an AR-CAG allele of >/=28, >/=29, or >/=30 repeats were given a diagnosis 0.8, 1.8, or 6.3 years earlier than women who did not carry at least one such allele. All 11 women in our sample who carried at least one AR-CAG allele with >/=29 repeats had breast cancer. Our results support the hypothesis that age at breast cancer diagnosis is earlier among BRCA1 mutation carriers who carry very long AR-CAG repeats. These results suggest that pathways involving androgen signaling may affect the risk of BRCA1-associated breast cancer.     

Systemic NF-kappaB activation in blood cells of breast cancer patients

There is a well-established role for reactive oxygen and nitrogen species, chronic inflammation and immune response in the pathogenesis of breast cancer. Complex interactions between breast cancer cells and surrounding blood vessels are prerequisites for cancer growth and invasion. Reports in the literature concerning the systemic response to, and the effect of, common breast cancer therapy on NF-kappaB and antioxidative defence enzyme expression and activity under clinical conditions are scarce. We determined these parameters in whole blood cell lysate from 16 women with breast cancer before and after combined (cyclophosphamide, doxorubicin, 5-fluorouracil; CAF) therapy and compared the results with 16 healthy women. Significantly higher levels of NF-kappaB and Mn-SOD (both their protein level and their activity) were found in breast cancer patients before and after CAF therapy, in comparison with healthy women. In parallel measurements, no change in the level or activity of catalase (CAT) was detected. According to our findings, it appears that breast cancer creates conditions that increase the level of hydrogen peroxide in the circulating cells and that the applied CAF therapy fails to compensate, therefore creating systemic conditions that favour survival and invasion of breast cancer cells.     

Breast cancer is marked by specific,Public T-cell receptor CDR3 regions shared by mice and humans

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.     

Influence of postmenopausal hormone replacement therapy on an estrogen metabolite biomarker of risk for breast cancer.

Whether postmenopausal hormone-replacement therapy (HRT) increases the risk of breast cancer remains controversial, despite numerous epidemiological studies. We approached the question from a biochemical rather than an epidemiological direction - we hypothesized that if estrogen administration increases the risk of breast cancer, it should also alter a known estrogen biomarker of risk towards what has been observed in patients who already have breast cancer. The specific biomarker we studied was the ratio of the urinary excretion of two principal estradiol metabolites, 2-hydroxyestrone and 16 alpha-hydroxyestrone, which is markedly decreased in women with breast cancer and women with familial risk for breast cancer. We studied 34 healthy postmenopausal women not on HRT and 19 women on HRT (Premarin 0.625 mg daily plus Provera, 2.5 mg daily, in women with a uterus and Premarin alone in women without a uterus); treatment duration ranged from 3 months to 15 years. We also studied four women with recently diagnosed, untreated breast cancer. The women with breast cancer showed a significantly lower 2-hydroxyestrone to 16 alpha-hydroxyestrone ratio than control women on HRT (1.35 +/- 0.13 vs. 2.71 +/- 0.84; p < 0.0001). There was no significant difference in the metabolite ratio between healthy women on HRT and women not on HRT (2.82 +/- 0.92 vs. 2.71 +/- 0.84). There was no significant difference between women receiving Premarin alone and women receiving Premarin plus Provera (2.46 +/- 0.84 vs. 3.13 +/- 0.90), and neither differed significantly from women not on HRT (2.71 +/- 0.84). The finding that the ratio of women on HRT was not decreased to or toward the ratio in women with breast cancer can be interpreted, we believe, as a suggestive item of biochemical evidence that HRT is not a risk for breast cancer.     

Protective properties of n-3 fatty acids and implications in obesity-associated breast cancer

Obesity is well documented as a risk factor for developing breast cancer, especially in postmenopausal women. Adipose tissue in the breast under obese conditions induces inflammation by increasing macrophage infiltration and pro-inflammatory cytokines that in turn up-regulates genes and signaling pathways, resulting in increased inflammation, cell proliferation and tumor growth in the breast. Due to their potent anti-inflammatory effects, n-3 polyunsaturated fatty acids (n-3 PUFA) are a promising and safe dietary intervention in reducing breast cancer risk. Here, we briefly review current status of breast cancer and its relationship with obesity. We then review in depth, current research and knowledge on the role of n-3 PUFA in reducing/preventing breast cancer cell growth in vitro, in vivo and in human studies, and how n-3 PUFA may modulate signaling pathways mitigating their effects on breast cancer development.     

Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2

Breast cancer remains the major cause of cancer-related deaths in women world-wide. The heterogeneity of breast cancer has further complicated the progress of target-based therapies. Triple negative breast cancers, lacking estrogen receptor, progesterone receptor and the Her-2/neu (ErbB2), represent a highly aggressive breast cancer subtype, that are difficult to treat. Pleiotropic agents, such as those found in nature, can target receptor-positive as well as receptor-negative cancer cells, suggesting that such agents could have significant impact in breast cancer prevention and/or therapy. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is one such agent which has anti-tumor activity against several cancers. However, its mechanism of action against breast cancer is not clearly understood. We hypothesized that plumbagin may act as an effective agent against breast cancer especially triple negative breast cancer. We tested our hypothesis using ER-positive MCF-7 and ER-negative MDA-MB-231 (triple negative) breast cancer cells, and we found that plumbagin significantly inhibits the growth of breast cancer cells with no effect on normal breast epithelial cells. We also found that plumbagin induces apoptosis with concomitant inactivation of Bcl-2 and the DNA binding activity of NF-kappaB. Bcl-2 over-expression resulted in attenuation of plumbagin-induced effects, suggesting that the inhibition of cell growth and induction of apoptosis by plumbagin is in part due to inactivation of NF-kappaB/Bcl-2 pathway. To our knowledge, this is the first report, showing mechanistic and cancer cell specific apoptosis-inducing effects of plumbagin in breast cancer cells, suggesting the potential role of plumbagin in the prevention and/or treatment of breast cancer.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.     

Risk factors for breast cancer in women biopsied for benign breast disease: A nested case-control study

Aim: Women with a history of benign breast disease are at increased risk of subsequent breast cancer. However, few studies have examined whether established breast cancer risk factors other than histology are associated with an altered risk of breast cancer in women with benign breast disease. We used a nested case-control design within a large, multi-center cohort of women biopsied for benign breast disease (BBD) to estimate odds ratios for breast cancer in association with exposure to a range of personal and lifestyle factors. Methods: Cases were women biopsied for BBD who subsequently developed breast cancer; controls were individually matched to cases on center and age at diagnosis and were women biopsied for BBD who did not develop breast cancer in the same follow-up interval as that for the cases. After excluding women with prevalent breast cancer, 1357 records (661 case records and 696 records) were available for analysis. We used conditional logistic regression to obtain crude and multivariable-adjusted estimates of the association between specific factors and risk of breast cancer. Results: In multivariable analyses age at first live birth, number of pregnancies, and postmenopausal status were inversely associated with risk of breast cancer. The odds ratio for women with age at first birth <25 years and ≥3 pregnancies, relative to nulliparous women, was 0.49, 95% confidence interval 0.13–0.79, and that for postmenopausal women relative to premenopausal women was 0.60, 95% CI 0.37–0.99. Conclusions: Further study of personal factors influencing the risk of breast cancer in women with BBD may help to identify subgroups of the population at increased risk of invasive disease.     

Thyroid and other autoantibodies in British and Japanese women: an epidemiological study of breast cancer.

To define the role of asymptomatic autoimmune thyroiditis in the cause of breast cancer, the presence of circulating thyroid autoantibodies was studied in two populations, one with a high risk of breast cancer (British women) and one with a low risk (Japanese women). Ostensibly healthy women and patients with breast cancer from both countries were studied. There was no difference in the incidence of thyroid autoantibodies between women with breast cancer and healthy women in either race. The incidence of thyroid autoantibodies in healthy British women, however, was two to three times that in healthy Japanese women. The incidence of reticulin antibodies, was considerably higher in both groups of Japanese women. No remarkable differences in the incidence of antinuclear, smooth-muscle, antimitochondrial, gastric parietal cell, or liver-kidney microsomal antibodies were found between women with breast cancer and healthy women or between the two races. Only the incidence of antinuclear antibodies was marginally higher in Japanese patients with advanced cancer. These results indicate that asymptomatic autoimmune thyroid disease is more prevalent among British than among Japanese women, but they fail to provide direct evidence that autoimmune thyroid disease is associated with breast cancer. Prospective studies of women with autoimmune thyroiditis and studies of young women from low-risk and high-risk populations are needed.     

Early-Life Socioeconomic Status and the Prevalence of Breast Cancer in Later Life

Knowledge of mechanisms linking early-life social environment and breast cancer remains limited. We explore direct and indirect effects of early-life socioeconomic status (SES) on breast cancer prevalence in later life. Using 50-year data from the Wisconsin Longitudinal Study (N = 4,275) and structural equation modeling, we found a negative direct effect of early-life SES, indicating that women from higher-SES family background had lower breast cancer prevalence than women from lower-SES families. Additionally, early-life SES has a positive indirect effect on breast cancer via women's adult SES and age at first birth. Were it not for their higher SES in adulthood and delayed childbearing, women from higher-SES families of origin would have had lower breast cancer prevalence than women from lower-SES families. Yet, early-life SES is associated positively with adult SES and age at first birth, and women's higher adult SES and delayed childbearing are related to higher breast cancer prevalence.     

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [³H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [³H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.     

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [³H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [³H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.     

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.     

Accumulation of Oxidatively Induced DNA Damage in Human Breast Cancer Cell Lines Following Treatment with Hydrogen Peroxide

Breast cancer is a leading cause of cancer deaths in women. Although the causes of this disease are largely unknown, inefficient repair of oxidatively induced DNA lesions has been thought to play a major role in the transformation of normal breast tissue to malignant breast tissue. Previous studies have revealed higher levels of 8-hydroxyguanine in malignant breast tissue compared to non-malignant breast tissue. Furthermore, some breast cancer cell lines have greatly reduced capacity to repair this lesion suggesting that oxidatively induced DNA lesions may be elevated in breast cancer cells. We used liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of 8-hydroxy-2’-deoxyadenosine, (5’S)-8,5’-cyclo-2’-deoxyadenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine in MCF-7 and HCC1937 breast cancer cell lines before and after exposure to H2O2 followed by a DNA repair period. We show that H2O2-treated HCC1937 and MCF-7 cell lines accumulate significantly higher levels of these lesions than the untreated cells despite a 1 h repair period. In contrast, the four lesions did not accumulate to any significant level in H2O2-treated non-malignant cell lines, AG11134 and HCC1937BL. Furthermore, MCF-7 and HCC1937 cell lines were deficient in the excision repair of all the four lesions studied. These results suggest that oxidatively induced DNA damage and its repair may be critical in the etiology of breast cancer.