Deciphering a proteomic signature for the early detection of breast cancer from breast milk: the role of quantitative proteomics.

INTRODUCTION: Breast cancer is one of the most prevalent cancers among women in the United States. Current research regarding breast milk has been focused on the composition and its role in infant growth and development. There is little information about the proteins, immune cells, and epithelial cells present in breast milk which can be indicative of the emergence of BC cells and tumors. AREAS COVERED: We summarize all breast milk studies previously done in our group using proteomics. These studies include 1D-PAGE and 2D-PAGE analysis of breast milk samples, which include within woman and across woman comparisons to identify dysregulated proteins in breast milk and the roles of these proteins in both the development of BC and its diagnosis. Our projected outlook for the use of milk for cancer detection is also discussed. EXPERT OPINION: Analyzing the samples by multiple methods allows one to interrogate a set of samples with various biochemical methods that complement each other, thus providing a more comprehensive proteome. Complementing methods like 1D-PAGE, 2D-PAGE, in-solution digestion and proteomics analysis with PTM-omics, peptidomics, degradomics, or interactomics will provide a better understanding of the dysregulated proteins, but also the modifications or interactions between these proteins.

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk.

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography-tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.

Prediagnostic breast milk DNA methylation alterations in women who develop breast cancer.

Prior candidate gene studies have shown tumor suppressor DNA methylation in breast milk related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis, we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer in two independent cohorts. DNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. Through an epigenome-wide association study we explored CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer using linear mixed effects models adjusted for history of breast biopsy, age, RefFreeCellMix cell estimates, time of delivery, array chip and subject as random effect. We identified 58 differentially methylated CpG sites associated with a subsequent breast cancer diagnosis (q-value <0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls. Breast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors and improving primary and secondary prevention of breast cancer.

New Moms Wellness Study: the randomized controlled trial study protocol for an intervention study to increase fruit and vegetable intake and lower breast cancer risk through weekly counseling and supplemental fruit and vegetable box delivery in breastfeeding women.

BACKGROUND: Laboratory studies indicate that chemicals in fruits and vegetables have anti-carcinogenic and anti-inflammatory activities that can lower breast cancer risk. However, epidemiologic studies of the association between fruit and vegetable intake and breast cancer risk have produced mixed results. Measurement error, confounding, and an emphasis on diet in later adulthood may contribute to weak associations. This paper describes a randomized controlled diet intervention trial in breastfeeding women to examine the effect of high fruit and vegetable intake on breast cancer risk factors, including weight, DNA methylation and inflammatory markers. METHODS: Eligible breastfeeding women who reside within a 35-mile radius of Amherst, MA are enrolled at five to six weeks postpartum and randomly assigned to a Fruit and Vegetable Intervention Arm (target n = 200) or to a USDA MyPlate Control Arm (target n = 200). The Fruit and Vegetable Intervention group receives weekly telephone or video-based counseling to encourage intake of at least eight to ten daily servings of fruits and vegetables and a weekly delivery of a supplemental box of fruits and vegetables for 20 weeks, and less intensive counseling for up to one year. Breastmilk and infant fecal specimens are collected at baseline, 10 and 20 weeks. Anthropometric measurements are obtained at these timepoints and at the 1-year follow-up. The primary outcomes are change in DNA methylation in breast epithelial cells and change in inflammatory markers in breastmilk from randomization to 20 weeks; and change in weight, waist circumference, and fruit and vegetable intake for the period from randomization to 20 weeks and 1 year. DISCUSSION: This 1-year randomized diet intervention trial in breastfeeding women will assess whether intake of at least eight to ten daily servings of fruits and vegetables per day improves biomarkers of breast cancer risk directly in the breast (i.e., DNA methylation and inflammatory markers) and helps women maintain a healthy weight. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04374747. Registered May 5, 2020. https://www. CLINICALTRIALS: gov/ct2/show/NCT04374747 .

Inter-Individual Variation in Response to Estrogen in Human Breast Explants.

Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10 nM 17ß-estradiol (E2) media for 4 days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFß2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.

Polymeric nanoassemblies for enrichment and detection of peptides and proteins in human breast milk.

Human breast milk is an understudied biological fluid that may be useful for early detection of breast cancer. Methods for enriching and detecting biomarkers in human breast milk, however, are not as well-developed as compared with other biological fluids. In this work, we demonstrate a new enrichment method based on polymeric nanoassemblies that is capable of enhancing the mass spectrometry-based detection of peptides and proteins in human breast milk. In this method, positively charged nanoassemblies are used to selectively deplete abundant proteins in milk based on electrostatic interactions, which simplifies the mixture and enhances detection of positively charged peptides and proteins. Negatively charged nanoassemblies are used in a subsequent enrichment step to further enhance the detection and quantification of trace-level peptides and proteins. Together the depletion and enrichment steps allow model biomarkers to be detected at low nM levels, which are close to instrumental limits of detection. This new method not only demonstrates the ability to detect proteins in human breast milk but also provides an alternative approach for targeted protein detection in complex biological matrices. Graphical abstract.

DNA methylation in breast cancers: Differences based on estrogen receptor status and recurrence.

DNA methylation plays a role in the etiology of primary breast cancers. We analyzed paired primary and second breast tumors to elucidate the role of methylation in recurrence. Methylation profiles from paired primary and second breast tumors of 23 women were assessed using the HumanMethylation450 BeadChip. Twelve women had estrogen receptor positive (ERpos) primary and second tumors, five had estrogen receptor negative (ERneg) primary and second tumors, and six had an ERpos primary tumor but an ERneg second tumor. Stratifying tumors by occurrence revealed that the greater methylation previously associated with ERpos tumors, is more pronounced in primary tumors than in second tumors. Further, ERneg second tumors are more methylated than ERpos second tumors among women who had ERpos primary tumors. Pathway analyses using gene lists generated from comparisons of methylation in ERpos primary tumors from the paired sets with ERpos tumors from six women without recurrences, identified differences between groups based on the ER status of the second tumor. Hypermethylated genes of significantly enriched pathways were differentially associated with survival. DNA methylation profiles of ERpos primary breast tumors support the development and use of tumor methylation profiles for stratifying women with breast cancer both for prognosis and therapy.

Combinatorial Electrophoresis and Mass Spectrometry-Based Proteomics in Breast Milk for Breast Cancer Biomarker Discovery.

Innovations in approaches for early detection and individual risk assessment of different cancers, including breast cancer (BC), are needed to reduce cancer morbidity and associated mortality. The assessment of potential cancer biomarkers in accessible bodily fluids provides a novel approach to identify the risk and/or onset of cancer. Biomarkers are biomolecules, such as proteins, that are indicative of an abnormality or a disease. Human milk is vastly underutilized biospecimen that offers the opportunity to investigate potential protein BC-biomarkers in young, reproductively active women. As a first step, we have examined the entire protein pattern in human milk samples from breastfeeding mothers with cancer, who were diagnosed either before or after milk donation, and from women without cancer, using mass spectrometry (MS)-based proteomics.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology.

BACKGROUND: Analysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors. METHODS: Participants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes. RESULTS: Thirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1ß, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1ß, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways. CONCLUSION: This exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

Proteomics analysis of human breast milk to assess breast cancer risk.

Detection of breast cancer (BC) in young women is challenging because mammography, the most common tool for detecting BC, is not effective on the dense breast tissue characteristic of young women. In addition to the limited means for detecting their BC, young women face a transient increased risk of pregnancy-associated BC. As a consequence, reproductively active women could benefit significantly from a tool that provides them with accurate risk assessment and early detection of BC. One potential method for detection of BC is biochemical monitoring of proteins and other molecules in bodily fluids such as serum, nipple aspirate, ductal lavage, tear, urine, saliva and breast milk. Of all these fluids, only breast milk provides access to a large volume of breast tissue, in the form of exfoliated epithelial cells, and to the local breast environment, in the form of molecules in the milk. Thus, analysis of breast milk is a non-invasive method with significant potential for assessing BC risk. Here we analyzed human breast milk by mass spectrometry (MS)-based proteomics to build a biomarker signature for early detection of BC. Ten milk samples from eight women provided five paired-groups (cancer versus control) for analysis of dysregulatedproteins: two within woman comparisons (milk from a diseased breast versus a healthy breast of the same woman) and three across women comparisons (milk from a woman with cancer versus a woman without cancer). Despite a wide range in the time between milk donation and cancer diagnosis (cancer diagnosis occurred from 1 month before to 24 months after milk donation), the levels of some proteins differed significantly between cancer and control in several of the five comparison groups. These pilot data are supportive of the idea that molecular analysis of breast milk will identify proteins informative for early detection and accurate assessment of BC risk, and warrant further research. Data are available via ProteomeXchange with identifier PXD007066.

Comparative two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of human milk to identify dysregulated proteins in breast cancer.

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC-MS/MS) analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.

TROP2 methylation and expression in tamoxifen-resistant breast cancer.

BACKGROUND: The DNA methyltransferase 1 inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-dC) is a potential treatment for breast cancer. However, not all breast tumors will respond similarly to treatment with 5-Aza-dC, and little is known regarding the response of hormone-resistant breast cancers to 5-Aza-dC. METHODS: We demonstrate that 5-Aza-dC-treatment has a stronger effect on an estrogen receptor-negative, Tamoxifen-selected cell line, TMX2-28, than on the estrogen receptor-positive, MCF7, parental cell line. Using data obtained from the HM450 Methylation Bead Chip, pyrosequencing, and RT-qPCR, we identified a panel of genes that are silenced by promoter methylation in TMX2-28 and re-expressed after treatment with 5-Aza-dC. RESULTS: One of the genes identified, tumor associated calcium signal transducer 2 (TACSTD2), is altered by DNA methylation, and there is evidence that in some cancers decreased expression may result in greater proliferation. Analysis of DNA methylation of TACSTD2 and protein expression of its product, trophoblast antigen protein 2 (TROP2), was extended to a panel of primary (n = 34) and recurrent (n = 34) breast tumors. Stratifying tumors by both recurrence and ER status showed no significant relationship between TROP2 levels and TACSTD2 methylation. Knocking down TACSTD2 expression in MCF7 increased proliferation however; re-expressing TACSTD2 in TMX2-28 did not inhibit proliferation, indicating that TACSTD2 re-expression alone was insufficient to explain the decreased proliferation observed after treatment with 5-Aza-dC. CONCLUSIONS: These results illustrate the complexity of the TROP2 signaling network. However, TROP2 may be a valid therapeutic target for some cancers. Further studies are needed to identify biomarkers that indicate how TROP2 signaling affects tumor growth and whether targeting TROP2 would be beneficial to the patient.

White blood cell DNA methylation and risk of breast cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO).

BACKGROUND: Several studies have suggested that global DNA methylation in circulating white blood cells (WBC) is associated with breast cancer risk. METHODS: To address conflicting results and concerns that the findings for WBC DNA methylation in some prior studies may reflect disease effects, we evaluated the relationship between global levels of WBC DNA methylation in white blood cells and breast cancer risk in a case-control study nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) cohort. A total of 428 invasive breast cancer cases and 419 controls, frequency matched on age at entry (55-59, 60-64, 65-69, ≥70 years), year of entry (on/before September 30, 1997, on/after October 1, 1997) and period of DNA extraction (previously extracted, newly extracted) were included. The ratio of 5-methyl-2' deoxycytidine [5-mdC] to 2'-deoxyguanine [dG], assuming [dG] = [5-mdC] + [2'-deoxycytidine [dC]] (%5-mdC), was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry, an especially accurate method for assessing total genomic DNA methylation. RESULTS: Odds ratio (OR) estimates and 95% confidence intervals (CI) for breast cancer risk adjusted for age at entry, year of entry, and period of DNA extraction, were 1.0 (referent), 0.89 (95% CI, 0.6-1.3), 0.88 (95% CI, 0.6-1.3), and 0.84 (95% CI, 0.6-1.2) for women in the highest compared to lowest quartile levels of %5md-C (p for trend = .39). Effects did not meaningfully vary by time elapsed from WBC collection to diagnosis. DISCUSSION: These results do not support the hypothesis that global DNA hypomethylation in WBC DNA is associated with increased breast cancer risk prior to the appearance of clinical disease.

Potential of breastmilk analysis to inform early events in breast carcinogenesis: rationale and considerations.

This review summarizes methods related to the study of human breastmilk in etiologic and biomarkers research. Despite the importance of reproductive factors in breast carcinogenesis, factors that act early in life are difficult to study because young women rarely require breast imaging or biopsy, and analysis of critical circulating factors (e.g., hormones) is often complicated by the requirement to accurately account for menstrual cycle date. Accordingly, novel approaches are needed to understand how events such as pregnancy, breastfeeding, weaning, and post-weaning breast remodeling influence breast cancer risk. Analysis of breastmilk offers opportunities to understand mechanisms related to carcinogenesis in the breast, and to identify risk markers that may inform efforts to identify high-risk women early in the carcinogenic process. In addition, analysis of breastmilk could have value in early detection or diagnosis of breast cancer. In this article, we describe the potential for using breastmilk to characterize the microenvironment of the lactating breast with the goal of advancing research on risk assessment, prevention, and detection of breast cancer.

ELF5 and DOK7 regulation in anti-estrogen treated cells and tumors.

BACKGROUND: Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. METHODS: Twenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 µM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women. RESULTS: All 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors. CONCLUSIONS: The distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.

Urinary melatonin concentration and the risk of breast cancer in Nurses' Health Study II.

Experimental and epidemiologic data support a protective role for melatonin in breast cancer etiology, yet studies in premenopausal women are scarce. In a case-control study nested within the Nurses' Health Study II cohort, we measured the concentration of melatonin's major urinary metabolite, 6-sulfatoxymelatonin (aMT6s), in urine samples collected between 1996 and 1999 among 600 breast cancer cases and 786 matched controls. Cases were predominantly premenopausal women who were diagnosed with incident breast cancer after urine collection and before June 1, 2007. Using multivariable conditional logistic regression, we computed odds ratios and 95% confidence intervals. Melatonin levels were not significantly associated with total breast cancer risk (for the fourth (top) quartile (Q4) of aMT6s vs. the first (bottom) quartile (Q1), odds ratio (OR) = 0.91, 95% confidence interval (CI): 0.64, 1.28; Ptrend = 0.38) or risk of invasive or in situ breast cancer. Findings did not vary by body mass index, smoking status, menopausal status, or time between urine collection and diagnosis (all Pinteraction values ≥ 0.12). For example, the odds ratio for total breast cancer among women with ≤5 years between urine collection and diagnosis was 0.74 (Q4 vs. Q1; 95% CI: 0.45, 1.20; Ptrend = 0.09), and it was 1.20 (Q4 vs. Q1; 95% CI: 0.72, 1.98; Ptrend = 0.70) for women with >5 years. Our data do not support an overall association between urinary melatonin levels and breast cancer risk.

Association of TGF-ß2 levels in breast milk with severity of breast biopsy diagnosis.

PURPOSE: TGF-ß plays a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-ß isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might be informative for breast cancer risk. Accordingly, we assessed TGF-ß2 levels in milk from women who had undergone a breast biopsy and related the concentrations to diagnosis. METHODS: Milk donated by women who had undergone or were scheduled for a breast biopsy was shipped on ice for processing and testing. Breast cancer risk factors were obtained through a self-administered questionnaire, and biopsy diagnoses were extracted from pathology reports. TGF-ß2 levels in milk, assessed as absolute levels and in relation to total protein, were analyzed in bilateral samples donated by 182 women. Linear regression was used to estimate relationships of log-transformed TGF-ß2 levels and TGF-ß2/ total protein ratios to biopsy category. RESULTS: Milk TGF-ß2 levels from biopsied and non-biopsied breasts within women were highly correlated (r (2) = 0.77). Higher mean TGF-ß2 milk levels (based on average of bilateral samples) were marginally associated with more severe breast pathological diagnosis, after adjusting for duration of nursing current child (adjusted p trend = 0.07). CONCLUSIONS: Our exploratory analysis suggests a borderline significant association between higher mean TGF-ß2 levels in breast milk and more severe pathologic diagnoses. Further analysis of TGF-ß signaling in milk may increase understanding of postpartum remodeling and advance efforts to analyze milk as a means of assessing risk of breast pathology.

SKP2 overexpression is associated with increased serine 10 phosphorylation of p27 (pSer10p27) in triple-negative breast cancer.

S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host's signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. RESULTS: In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatis infection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-ß (PDGFRß) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRß that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatis development. CONCLUSION: Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases.

Using breast milk to assess breast cancer risk: the role of mass spectrometry-based proteomics.

Although mammography and treatment advances have led to declines in breast cancer mortality in the United States, breast cancer remains a major cause of morbidity and mortality. Breast cancer in young women is associated with increased mortality and current methods of detecting breast cancers in this group of women have known limitations. Tools for accurately assessing personal breast cancer risk in young women are needed to identify those women who would benefit the most from earlier intervention. Proteomic analysis of breast milk could identify biomarkers of breast cancer risk and provide a tool for identifying women at increased risk. A preliminary analysis of milk from four women provides a proof of concept for using breast milk to assess breast cancer risk.