What is Breast Cancer?

Breast cancer is the most common tumour in women and the first cause of death for cancer in the female population. Preserving the quality of life has therefore become an important objective in the management of the disease. The benefits of adjuvant chemotherapy in patients with HR+ HER2- early breast cancer should always be balanced against its potential short and long-term adverse effects, and identifying the appropriate patients for whom chemotherapy can offer the highest clinical benefit is critical. Besides clinical and pathological factors, today four multigene tests able to guide the choice of the adjuvant therapy early breast cancer are available in Italy: Oncotype DX^®, EndoPredict^®, MammaPrint^® e Prosigna^®. This review evaluates the main characteristics of these diagnostic tests, the studies on clinical utility, their economic impact and their inclusion in international and national guidelines. The Oncotype DX Breast Recurrence Score^® test is the only multigene test validated, with level IA evidence, to guide the adjuvant therapy decisions: hormone therapy alone for most patients with RS results 0–25, and chemotherapy for patients with RS results 26–100. Clinical data demonstrate that the Oncotype DX test is able to significantly impact therapeutic decisions, reducing chemotherapy use up to 49% and supporting the use of chemotherapy (up to 12%) in potentially under-treated patients. Based on the level of clinical evidence and established clinical utility, several multigene tests have been included in the main international guidelines, with recommendations ranging from “strong” to “moderate”.

Vitamin C may impact the efficiency of radiation therapy (RT) in breast cancer. The effects of RT alone or in combination with vitamin C in SKBR3, MDA-MB-231, and MCF7 cells were compared using clonogenic assay, proliferation assay (MTT), cell cycle analysis, and Western blot. Vitamin C use was assessed in 1803 breast cancer patients 2002–2017 in relation to clinicopathological features and recurrences after RT. Vitamin C combined with RT resulted in non-significant increases in colony formation and minor differences in cell cycle arrest and expression of studied proteins, compared to RT alone. Lower vitamin C doses alone or in combination with RT, resulted in higher proliferation with MTT than higher vitamin C doses in a cell line-dependent manner. Vitamin C use was associated with lower histological grade and BMI but not recurrence risk in RT-treated patients (LogRank P = 0.54). Vitamin C impacted RT efficiency differently depending on breast cancer subtype and vitamin C concentration. Lower doses of vitamin C, achievable with oral administration, might increase breast cancer cell proliferation and decrease radiosensitivity. Despite vitamin C users having less aggressive tumors than non-users, the recurrence risk in RT-treated patients was similar in vitamin C users and non-users.

GZ17-6.02 is presently undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in estrogen receptor positive breast cancer cells and to determine whether it interacted with palbociclib to enhance tumor cell killing. GZ17-6.02 interacted in an additive fashion with palbociclib to kill ER+ breast cancer cells. GZ17-6.02 and palbociclib cooperated to inactivate mTOR and AKT and to activate ULK1 and PERK. The drugs interacted to increase the expression of FAS-L and BAX, and to decrease the levels of MCL1, the estrogen receptor, and HDACs 1–3. Palbociclib activated ERBB3, an effect blocked by GZ17-6.02. GZ17-6.02 and palbociclib interacted to increase the expression of multiple toxic BH3 domain proteins and to reduce MCL1 and BCL-XL expression. Knock down of FAS-L reduced the lethality of [GZ17-6.02 + palbociclib]. GZ17-6.02 and palbociclib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, BAG3, eIF2α, toxic BH3 domain proteins or CD95 significantly reduced drug combination lethality. GZ17-6.02 and palbociclib increased the expression of Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs also increased the phosphorylation of the AMPK and ATG13, effects blocked by knock down of ATM. Knock down of ATM or the AMPK, or expression of activated mTOR significantly reduced the abilities of GZ17-6.02 and palbociclib to enhance autophagosome formation and autophagic flux.

Breast cancer (BC) is the most common type of cancer diagnosed in women. Among female cancer deaths, BC is the second leading cause of death worldwide. For estrogen receptor-positive (ER-positive) breast cancers, endocrine therapy is an effective therapeutic approach. However, in many cases, an ER-positive tumor becomes unresponsive to endocrine therapy, and tumor regrowth occurs after treatment. While some genetic mutations contribute to resistance in some patients, the underlying causes of resistance to endocrine therapy are mostly undetermined. In this study, we utilized a recently developed statistical approach to investigate the dynamic behavior of gene expression during the development of endocrine resistance and identified a novel group of genes whose time course expression significantly change during cell modelling of endocrine resistant BC development. Expression of a subset of these genes was also differentially expressed in microarray analysis of endocrine-resistant and endocrine-sensitive tumor samples. Surprisingly, a subset of those genes was also differentially genes expressed in triple-negative breast cancer (TNBC) as compared with ER-positive BC. The findings suggest shared genetic mechanisms may underlie the development of endocrine resistant BC and TNBC. Our findings identify 34 novel genes for further study as potential therapeutic targets for treatment of endocrine-resistant BC and TNBC.

Highly adaptable breast cancer cells that can opportunistically switch between proliferation and quiescence are often responsible for disease relapse. We have developed a function-based selection strategy for such resistant cells, exemplified by SUM149-MA and FC-IBC02-MA triple-negative breast cancer cells. We have also reported that a lengthy treatment with low-dose 6-mercaptopurine, a clinically useful anti-inflammatory drug, inhibits such resistant cells. To more rigorously test the clinical suitability of 6-mercaptopurine, here we investigated effects of further lowering its dose and the possibility of overcoming resistance to single-drug treatment by combining the drug with another ribonucleoside analog 5-azacitidine. We found that that a lengthy treatment with 1 μM 5-azacitidine, without a significant effect on cell proliferation, sensitized cancer cells to the inhibitory effects of low-dose 6-mercaptopurine. Importantly, treatment for several weeks with low doses of 6-mercaptopurine and/or 5-azacitidine did not render cancer cells resistant to chemotherapeutic drugs doxorubicin or paclitaxel. In fact, the cells became more sensitive to chemotherapeutic drugs upon treatment with 6-mercaptopurine and/or 5-azacitidine. Our analyses of protein markers of epithelial-to-mesenchymal transition indicated that treatments with 6-mercaptopurine and/or 5-azacitidine do not significantly reverse this process in our model. Our results showed that safe drugs such as low-dose 6-mercaptopurine singly or combined with 5-azacitidine, which are suitable for use prior to disease relapse, have a potential of inhibiting highly resistant triple-negative breast cancer cells.

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

Breast cancer (BC) and colorectal cancer (CRC) are common and show poor survival in advanced stages. Using The Cancer Genome Atlas (TCGA) computational tool cBioPortal, we evaluated overall patient survival in BRCA1 mRNA-low versus -high cohorts (<−1.29 versus >1.05 SD from mean BRCA1 expression, respectively). Analysis included 1082 BC patients with mRNA data (PanCancer Atlas), 382 CRCs (Firehose Legacy) and 592 CRCs (PanCancer Atlas). As previously reported, BRCA1 mRNA-low tumor expression positively correlated with BC patient survival but was negatively associated in CRC. We observed a correlation between BRCA1 mRNA-high and age <45 years at CRC diagnosis using a Fisher’s exact test [Firehose Legacy database (p-value = 0.0091); CRC PanCancer Atlas (p-value = 0.0778)]. We correlated BRCA1 mRNA-low expression and basal BC (p-value = 0.0016) and BRCA1 mRNA-low tumors and frequency of African American patients (p-value = 0.0448) with BC. Other trends included higher frequency of advanced lymph node stage and mucinous adenocarcinoma among BRCA1 mRNA-low CRC and higher frequency of males in BRCA1 mRNA-high BC and CRC. African Americans more frequently had BRCA1 mRNA-low BC and BRCA1 mRNA-high CRC and the opposite was observed among Asians. Using a gene co-expression tool (cBioPortal), we observed TOP2A and ATAD5 levels correlate (Spearman’s correlation>0.6) with BRCA1 in BC and CRC, whereas LMNB2 correlates with BRCA1 in CRC, suggesting tissue-specific BRCA1 interactions. Our results indicate potential for BRCA1 mRNA expression levels as a prognostic biomarker in BC and CRC, suggest tissue-specificity in BRCA1 molecular interactions, and point to BRCA1 mRNA-high levels as a characteristic of CRC tumors in younger versus older individuals.

Neutrophils are prominent immune components of tumors, having either anti-tumor (N1) or pro-tumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN are rare in non-pathological conditions, but frequent in cancer, exhibiting a pro-tumor phenotype. These findings have been mainly demonstrated in animal models, thus proper validation in humans is still imperative. Here, we observed that LDN were increased in the blood of breast cancer (BC) patients, particularly with metastatic disease. Within the population of non-metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than patients with a good response. The higher incidence of LDN in BC patients with severe disease or resistance to treatment can be explained by their pro-tumor/immunosuppressive characteristics. Moreover, the percentage of LDN in BC patients’ blood was negatively correlated with activated cytotoxic T lymphocytes and positively correlated with immunosuppressive regulatory T cells. The ability of LDN to spoil anti-tumor immune responses was further demonstrated ex vivo. Hence, this study reveals the potential of LDN as a biomarker of BC response to treatment and opens new avenues for developing new immunotherapies.

Background: Tumor protein 53 (TP53) gene mutations are identified in up to 37% of breast tumors especially in HER-2 positive and basal-like subtype. Previous studies have indicated TP53 mutations as a prognostic biomarker in breast cancer. However, most of these studies performed immunohistochemistry (IHC) for the detection of TP53 mutations.

Aim: The purpose of our study is to evaluate the role of TP53 somatic mutations detected via next-generation sequencing (NGS) as a potential prognostic marker in patients with breast cancer.

Materials and Methods: 82 female patients with Stage I–III breast cancer underwent NGS in paraffin blocks and blood samples during the period 25/09/2019 through 25/05/2021. 23 cases of somatic TP53 mutations and 23 cases of healthy controls were matched on age at diagnosis, menopausal status, histological subtype, histological grade, ki67 expression and disease stage.

Results: Mean age at diagnosis was 52.35 (SD; 11.47) years. The somatic TP53 mutation NM_000546.5:c.824G>A p.(Cys275Tyr) was most frequently detected. Co-existence of PIK3CA mutation was a common finding in somatic TP53-mutant tumors (4/23; 17.4%). Disease-free survival was shorter in TP53-mutated cases (16.3 months vs. 62.9 months). TP53 pathogenic somatic mutations were associated with a 8-fold risk of recurrence in the univariate Cox regression analysis (OR = 8.530, 95% CI: 1.81–40.117; p = 0.007).

Conclusions: Our case-control study suggests that TP53 somatic mutations detected by next-generation sequencing (NGS) are associated with an adverse prognosis in breast cancer.

LKB1-signaling has prominent roles in cancer development and metastasis. This report evaluates LKB1-signaling pathway gene expression associations with patient survival in overall breast cancer, specific subtypes, as well as pre- and post-chemotherapy. Subtypes analyzed were based on intrinsic molecular subtyping and traditional biomarker classifications. Intrinsic molecular subtypes included were Luminal-A, Luminal-B, HER2-enriched, and Basal-like. The biomarker subtypes assessed were Estrogen-Receptor Positive (ER+) and Negative (ER-), Wild-Type TP53 (WT-TP53) & Mutant-TP53, and Triple-Negative Breast Cancer (TNBC). Additionally, comparisons were made between these subtypes and breast cancer overall, and analyses between LKB1 signaling to patient survival before and after chemotherapy were made. We used the Kaplan-Meier Online Tool (KM Plotter) to correlate the relationship between mRNA expression of known LKB1 scaffolding proteins (CAB39 and LYK5), and downstream signaling targets (AMPK, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, PAK1, SIK1, SIK2, BRSK1, BRSK2, SNRK, and QSK), and patient survival across each subtype and treatment group. Our findings provide evidence that LKB1-signaling is associated with improved survival in overall breast cancer. Stratification into breast cancer subtypes show a more complicated relationship; NUAK2, for example, is correlated with improved survival in ER- but is worse in ER+ breast cancer. In evaluating the association of LKB1-signaling pathway expression with relapse free survival of varying breast cancer tumors exposed to chemotherapy or treatment-naive tumors, our data provides baseline knowledge for understanding the pathway dynamics that affect survival and therefore are linked to pathology. This establishes a foundation for studying LKB1 targets with the goal of identifying druggable targets.

Plasma based genotyping via cell-free DNA may identify actionable mutations for potential therapeutic intervention in patients with advanced malignancies including breast cancer. In this article, we discuss recent studies using cell-free DNA testing to identify and classify somatic BRCA1/2 mutations in metastatic breast cancer, and potential future applications for the treatment of metastatic breast cancer.

Most patients with early HR+ and HER2- breast cancer receive a hormone therapy; the clinical question still open is how to identify patients who can really benefit from adjuvant chemotherapy. The accurate identification of these patients is essential to avoid an over-treatment, increasing the risk of an unnecessary toxicity; on the contrary, the omission of chemotherapy can deprive high risk patients of a potential life-saving treatment (under-treatment). Several multigene assays (MGAs), assessing the risk of relapse according to the biological characteristics of the tumor, have been developed. To date, the 21-gene assay (Oncotype DX Breast Recurrence Score^®) is the only test developed and validated to be actionable, i.e., able to predict the benefit of adjuvant chemotherapy. The different available tests can be classified according to their clinical utility based on their prognostic and predictive value. A prognostic test gives information about the outcome of the disease, regardless of the administered therapy. When the aim of the test is to drive the treatment decisions, the predictive component, and therefore the ability to accurately identify which patients could benefit from chemotherapy, is essential. This review summarizes the clinical evidences of the Oncotype DX^® test supporting its clinical utility.

Breast cancer (BC) in Nigeria is characterized by disproportionately aggressive molecular subtypes. C-reactive protein (CRP) is associated with risk and aggressiveness for several types of cancer. We examined the association of high-sensitivity CRP (hsCRP) with odds of BC by molecular subtype among Nigerian women. Among 296 newly diagnosed BC cases and 259 healthy controls, multivariable logistic regression models were used to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI) for the association between hsCRP and odds of BC overall and by molecular subtype (luminal A, luminal B, HER2-enriched and triple-negative or TNBC). High hsCRP (> 3 mg/L) was observed in 57% of cases and 31% of controls and was associated with 4 times the odds of BC (aOR: 4.43; 95% CI: 2.56, 7.66) after adjusting for socio-demographic, reproductive, and clinical variables. This association persisted regardless of menopausal status and body mass index (BMI) category. High hsCRP was associated with increased odds of TNBC (aOR: 3.32; 95% CI: 1.07, 10.35), luminal A BC (aOR: 4.03; 95% CI: 1.29, 12.64), and HER2-enriched BC (aOR: 6.27; 95% CI: 1.69, 23.25). Future studies are necessary in this population to further evaluate a potential role for CRP as a predictive biomarker for BC.

Breast cancer (BCa) ranks first in incidence rate among cancers in Arab females. The association between genetic polymorphisms in tumor suppressor genes and the risk of BCa has been studied in many ethnic populations with conflicting conclusions while Arab females and Saudi Arabian studies are still lacking. We screened a cohort of Saudi BCa patients by NGS using a bespoke gene panel to clarify the genetic landscape of this population, correlating and assessing genetic findings with clinical outcomes. We identified a total of 263 mutations spanning 51 genes, including several frequently mutated. Among the genes analyzed, the highest mutation rates were found in PIK3CA (12.9%), BRCA2 (11.7%), BRCA1 (10.2%), TP53 (6.0%), MSH2 (3.8%), PMS2 (3.8%), BARD1 (3.8%), MLH1 (3.4%), CDH1 (3.0%), RAD50 (3.0%), MSH6 (3.0%), NF1 (2.6%), in addition to others. We identified multiple common recurrent variants and previously reported mutations. We also identified 46 novel variants in 22 genes that were predicted to have a pathogenic effect. Survival analysis according to the four most common mutations (BRCA1, BRCA2, TP53, and PIK3CA) showed reduced survival in BRCA1 and BRCA2-mutant patients compared to total patients. Moreover, BRCA2 was demonstrated as an independent predictor of reduced survival using independent Cox proportional hazard models.

We reveal the landscape of the mutations associated with BCa in Saudi women, highlighting the importance of routine genetic sequencing in implementation of precision therapies in KSA.

Tumor mutational burden (TMB) is a promising tool to help define patients with triple-negative breast cancer (TNBC) most likely to benefit from immune checkpoint blockade (ICB) therapies. Roughly reflecting the degree of neo-antigens that tumors present to immune cells, TMB associates with multiple measures of tumoral immunogenicity and has proven clinically useful in cancers with relatively high mutation burden. TNBC carries higher TMB than other breast cancer subtypes, and recent data suggest that high-TMB TNBC cases may derive particular benefit from ICB in combination with chemotherapy (GeparNuevo, IMpassion130) or even ICB alone (KEYNOTE-119, TAPUR). Given the recent approval of pembrolizumab and atezolizumab in combination with chemotherapy for PD-L1-positive, metastatic TNBC, standardizing TMB calculation methods and cut-off values is of critical importance to deploy this clinical biomarker.

Background: The purpose of the study was to investigate the role of pre-treatment quantitative ultrasound (QUS)-radiomics in predicting recurrence for patients with locally advanced breast cancer (LABC).

Materials and Methods: A prospective study was conducted in patients with LABC (n = 83). Primary tumours were scanned using a clinical ultrasound device before starting treatment. Ninety-five imaging features were extracted-spectral features, texture, and texture-derivatives. Patients were determined to have recurrence or no recurrence based on clinical outcomes. Machine learning classifiers with k-nearest neighbour (KNN) and support vector machine (SVM) were evaluated for model development using a maximum of 3 features and leave-one-out cross-validation.

Results: With a median follow up of 69 months (range 7–118 months), 28 patients had disease recurrence (local or distant). The best classification results were obtained using an SVM classifier with a sensitivity, specificity, accuracy and area under curve of 71%, 87%, 82%, and 0.76, respectively. Using the SVM model for the predicted non-recurrence and recurrence groups, the estimated 5-year recurrence-free survival was 83% and 54% (p = 0.003), and the predicted 5-year overall survival was 85% and 74% (p = 0.083), respectively.

Conclusions: A QUS-radiomics model using higher-order texture derivatives can identify patients with LABC at higher risk of disease recurrence before starting treatment.

Over the last decade, breast cancer mortality has declined. However, triple negative breast cancer (TNBC) remains a challenging problem mostly due to early recurrence and lack of molecularly driven treatments. There is a critical need to identify subgroups of TNBC with common molecular features that can be therapeutically targeted. Here we show that in contrast to Klotho and βKlotho, the third member of the Klotho protein family, γKlotho, is overexpressed in more than 60% of TNBCs and correlates with poorer disease progression. Furthermore, we find that γKlotho is expressed in a subset of TNBC cell lines promoting cell growth. Importantly, we demonstrate that in these cells γKlotho is necessary for cell survival and that its depletion leads to constitutive ERK activation, cell cycle arrest and apoptosis. Interestingly, we observe increased oxidative stress in γKlotho-depleted cells suggesting that γKlotho enables cancer cells to cope with an oxidative environment and that cells become dependent on its expression to maintain this survival advantage. These findings indicate that γKlotho might be a potential marker for patients that would benefit from treatments that alter oxidative stress and constitutes a novel drug target for a subset of TN breast cancers.

The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways.

Werner syndrome protein (WRN) is a RecQ helicase that participates in DNA repair, genome stability and cellular senescence. The five human RecQ helicases, RECQL1, Bloom, WRN, RECQL4 and RECQL5 play critical roles in DNA repair and cell survival after treatment with the anticancer drug camptothecin (CPT). CPT derivatives are widely used in cancer chemotherapy to inhibit topoisomerase I and generate DNA double-strand breaks during replication. Here we studied the effects of CPT on the stability and expression dynamics of human RecQ helicases. In the cells treated with CPT, we observed distinct effects on WRN compared to other human RecQ helicases. CPT altered the cellular localization of WRN and induced its degradation by a ubiquitin-mediated proteasome pathway. WRN knockdown cells as well as CPT treated cells became senescent and stained positive for senescence-associated β-galactosidase at a higher frequency compared to control cells. However, the senescent phenotype was attenuated by ectopic expression of WRN suggesting functional implication of WRN degradation in CPT treated cells. Approximately 5-23% of breast cancer tumors are known to respond to CPT-based chemotherapy. Interestingly, we found that the extent of CPT-induced WRN degradation correlates with increasing sensitivity of breast cancer cells to CPT. The abundance of WRN decreased in CPT-treated sensitive cells; however, WRN remained relatively stable in CPT-resistant breast cancer cells. In a large clinical cohort of breast cancer patients, we find that WRN and topoisomerase I expression correlate with an aggressive tumor phenotype and poor prognosis. Our novel observations suggest that WRN abundance along with CPT-induced degradation could be a promising strategy for personalizing CPT-based cancer chemotherapeutic regimens.

Osteoprotegerin (OPG) is a soluble decoy receptor for tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). It belongs to the tumor necrosis factor receptor superfamily (TNFRSF). OPG was initially discovered to contribute to homeostasis of bone turnover due to its capability of binding to receptor activator of nuclear factor-kappaB (NF-kB). However, apart from bone turnover, OPG plays important and diverse role(s) in many biological functions. Besides having anti-osteoclastic activity, OPG is thought to exert a protective anti-apoptotic action in OPG-expressing tumors by overcoming the physiologic mechanism of tumor surveillance exerted by TRAIL. Along with inhibiting TRAIL induced apoptosis, it can induce proliferation by binding to various cell surface receptors and thus turning on the canonical cell survival and proliferative pathways. OPG also induces angiogenesis, one of the hallmarks of cancer, thus facilitating tumor growth. Recently, the understanding of OPG and its different roles has been augmented substantially. This review is aimed at providing a very informative overview as to how OPG affects cancer progression especially breast cancer.

Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

Background: TP53 mutations are frequent in breast cancer, however their clinical relevance in terms of response to chemotherapy is controversial.

Methods: 450 pre-therapeutic, formalin-fixed, paraffin-embedded core biopsies from the phase II neoadjuvant GeparSixto trial that included HER2-positive and triple negative breast cancer (TNBC) were subjected to Sanger sequencing of exons 5-8 of the TP53 gene. TP53 status was correlated to response to neoadjuvant anthracycline/taxane-based chemotherapy with or without carboplatin and trastuzumab/lapatinib in HER2-positive and bevacizumab in TNBC. p53 protein expression was evaluated by immunohistochemistry in the TNBC subgroup.

Results: Of 450 breast cancer samples 297 (66.0%) were TP53 mutant. Mutations were significantly more frequent in TNBC (74.8%) compared to HER2-positive cancers (55.4%, P < 0.0001). Neither mutations nor different mutation types and effects were associated with pCR neither in the whole study group nor in molecular subtypes (P > 0.05 each). Missense mutations tended to be associated with a better survival compared to all other types of mutations in TNBC (P = 0.093) and in HER2-positive cancers (P = 0.071). In TNBC, missense mutations were also linked to higher numbers of tumor-infiltrating lymphocytes (TILs, P = 0.028). p53 protein overexpression was also linked with imporved survival (P = 0.019).

Conclusions: Our study confirms high TP53 mutation rates in TNBC and HER2-positive breast cancer. Mutations did not predict the response to an intense neoadjuvant chemotherapy in these two molecular breast cancer subtypes.

There are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are functional candidates for further investigation as disease-causing variants. To investigate whether common variants associated with differential allelic expression were involved in breast cancer susceptibility, a list of genes was established on the basis of their involvement in cancer related pathways and/or mechanisms. Thereafter, using data from a genome-wide map of allelic expression associated SNPs, 313 genetic variants were selected and their association with breast cancer risk was then evaluated in 46,451 breast cancer cases and 42,599 controls of European ancestry ascertained from 41 studies participating in the Breast Cancer Association Consortium. The associations were evaluated with overall breast cancer risk and with estrogen receptor negative and positive disease. One novel breast cancer susceptibility locus on 4q21 (rs11099601) was identified (OR = 1.05, P = 5.6x10-6). rs11099601 lies in a 135 kb linkage disequilibrium block containing several genes, including, HELQ, encoding the protein HEL308 a DNA dependant ATPase and DNA Helicase involved in DNA repair, MRPS18C encoding the Mitochondrial Ribosomal Protein S18C and FAM175A (ABRAXAS), encoding a BRCA1 BRCT domain-interacting protein involved in DNA damage response and double-strand break (DSB) repair. Expression QTL analysis in breast cancer tissue showed rs11099601 to be associated with HELQ (P = 8.28x10-14), MRPS18C (P = 1.94x10-27) and FAM175A (P = 3.83x10-3), explaining about 20%, 14% and 1%, respectively of the variance inexpression of these genes in breast carcinomas.

Long non-coding RNAs (lncRNAs) have been implicated in normal cellular homeostasis as well as pathophysiological conditions, including cancer. Here we performed global gene expression profiling of mammary epithelial cells transformed by oncogenic v-Src, and identified a large subset of uncharacterized lncRNAs potentially involved in breast cancer development. Specifically, our analysis revealed a novel lncRNA, LINC00520 that is upregulated upon ectopic expression of oncogenic v-Src, in a manner that is dependent on the transcription factor STAT3. Similarly, LINC00520 is also increased in mammary epithelial cells transformed by oncogenic PI3K and its expression is decreased upon knockdown of mutant PIK3CA. Additional expression profiling highlight that LINC00520 is elevated in a subset of human breast carcinomas, with preferential enrichment in the basal-like molecular subtype. ShRNA-mediated depletion of LINC00520 results in decreased cell migration and loss of invasive structures in 3D. RNA sequencing analysis uncovers several genes that are differentially expressed upon ectopic expression of LINC00520, a significant subset of which are also induced in v-Src-transformed MCF10A cells. Together, these findings characterize LINC00520 as a lncRNA that is regulated by oncogenic Src, PIK3CA and STAT3, and which may contribute to the molecular etiology of breast cancer.

Interleukin-12 (IL-12) is a proinflammatory cytokine that links innate and adaptive immune responses against tumor cells. Single Nucleotide Polymorphisms (SNPs) in IL-12 genes have been associated with cancer risk. However, limited studies have assessed the role of IL-12 in breast cancer (BC) risk comprehensively, and these were done in European and Asian populations. Here, we evaluated the association of the IL-12 signaling pathway and BC risk in Puerto Rican women. A genetic association study was completed with 461 BC cases and 463 non-BC controls. By logistic regression, IL-12 signaling SNPs were associated with an increased BC risk, including rs2243123 (IL12A), rs3761041, rs401502 and rs404733 (IL12RB1), rs7849191 (JAK2), rs280500 (TYK2) and rs4274624 (STAT4). Conversely, other SNPs were associated with reduced BC risk including rs438421 (IL12RB1), rs6693065 (IL12RB2), rs10974947, and rs2274471 (JAK2), rs10168266 and rs925847 (STAT4), and rs2069718 (IFNG). Analyses based in hormone receptors such as estrogen (ER) and progesterone (PR) receptors also revealed protective (for SNPs rs3212227-IL12B; rs3024896 and rs3821236-STAT4) and predisposing (for rs2069705-IFNG SNP) BC associations. Haplotype analysis showed a decreased BC risk for IL12B and STAT4 SNPs, whereas increased risk for IL12RB1 SNPs. This study suggests a role of the IL-12 signaling axis and BC risk. SNPs in this pathway may alter IL-12 induced anti-tumor responses and modulate BC predisposition in a population-specific context. Functional studies will be necessary to confirm these findings, which potentially may benefit IL-12 related immunotherapeutic approaches towards BC.

Purpose: High throughput panel sequencing to tailor therapy in precision oncology promises to improve outcome in patients with metastatic breast cancer. However, data that clearly show any benefit from such an approach is still pending.

Materials and Methods: We performed a retrospective analysis of advanced breast cancer patients that underwent panel sequencing for suggestion of target related drugs. We aimed to (i) determine the frequency of actionable mutations per patient and to (ii) assess the clinical impact of results on treatment options.

Results: A total of 52 patients underwent panel sequencing of archived tumor tissue. Every sample showed at least one affected gene, accounting for actionable mutations in 45 of 52 patients (87%). New treatment options that would not have been used as indicated by standard predictive markers (such as hormonal receptor status or HER2-status) were found in 22 of 52 patients (42%). We detected therapeutic relevant pathogenic germline variants in 9,6% (5/52) of the patients.

Conclusions: Using a high throughput-panel sequencing approach to identify actionable mutations in patients with metastatic breast cancer, we identified potential target-related treatment options in a large proportion of our patients, some of which would not have been considered without this data. Prospective clinical trials with compounds targeting the identified actionable mutations are needed to determine which treatments can indeed improve survival or quality of life by limiting exposure to ineffective drugs in advanced breast cancer.

The influence of breast cancer cells on normal cells of the microenvironment, such as fibroblasts and macrophages, has been heavily studied but the influence of normal epithelial cells on breast cancer cells has not. Here using in vivo and in vitro models we demonstrate the impact epithelial cells and the mammary microenvironment can exert on breast cancer cells. Under specific conditions, signals that originate in epithelial cells can induce phenotypic and genotypic changes in cancer cells. We have termed this phenomenon “cancer cell redirection.” Once breast cancer cells are redirected, either in vivo or in vitro, they lose their tumor forming capacity and undergo a genetic expression profile shift away from one that supports a cancer profile towards one that supports a non-tumorigenic epithelial profile. These findings indicate that epithelial cells and the normal microenvironment influence breast cancer cells and that under certain circumstances restrict proliferation of tumorigenic cells.

Targeted drug approaches have been a major focus for developing new anticancer therapies. Although many such agents approved in the last 20 years have improved outcomes, almost all have underperformed expectations. The full potential of such agents may yet be obtained through novel combinations. Previously, we showed that anti-estrogen drugs combined with a dendritic cell-based anti-HER-2 vaccine known to induce strong Th1-polarized immunity dramatically improved clinical response rates in patients with HER-2^pos/ER^pos early breast cancer. Here, we show that the small molecule Akt antagonist MK-2206, when combined with the Th1 cytokines IFN-gamma and TNF-alpha, maximize indicators of apoptotic cell death in a panel of phenotypically-diverse human breast cancer lines. These findings were mirrored by other, structurally-unrelated Akt-targeting drugs that work through different mechanisms. Interestingly, we found that MK-2206, as well as the other Akt antagonist drugs, also had a tendency to suppress Th1 cytokine expression in stimulated human and murine lymphocytes, potentially complicating their use in conjunction with active immunotherapy. After verifying that MK-2206 plus IFN-gamma could show similar combined effects against breast cancer lines, even in the absence of TNF-alpha, we tested in a rodent HER-2^pos breast cancer model either a HER-2-based DC vaccine, or recombinant IFN-gamma with or without MK-2206 administration. We found that for MK-2206, co-administration of recombinant IFN-gamma outperformed co-administration of DC vaccination for slowing tumor growth kinetics. These findings suggest a combined therapy approach for Akt-targeting drugs that incorporates recombinant Interferon-gamma and is potentially translatable to humans.

1,25-Dihydroxyvitamin D₃ (1,25D3) induces growth arrest and apoptosis in breast cancer cells in vivo and in vitro, however the exact mechanisms are unclear. Although the vitamin D receptor (VDR), a ligand dependent transcription factor, is required for growth regulation by vitamin D, the specific target genes that trigger these effects are unknown. Genomic profiling of murine mammary tumor cells with differential VDR expression identified 35 transcripts that were altered by the 1,25D3-VDR complex including Hyaluronan Synthase-2 (Has2). Here we confirmed that 1,25D3 reduces both HAS2 gene expression and hyaluronic acid (HA) synthesis in multiple models of breast cancer. Furthermore, we show that the growth inhibitory effects of 1,25D3 are partially reversed in the presence of high molecular weight HA. HAS2 expression and HA production are elevated in immortalized human mammary epithelial cells induced to undergo epithelial-mesenchymal transition (EMT) through stable expression of TGFβ, SNAIL or TWIST and in those expressing oncogenic H-RAS^V12, indicating that deregulation of HA production may be an early and frequent event in breast tumorigenesis. 1,25D3 also reduces HA secretion and acts additively with an HA synthesis inhibitor to slow growth of cells expressing TGFβ, SNAIL and TWIST. Analysis of mammary gland and tumors from Vdr knockout mice suggest that loss of VDR is associated with enhanced HAS2 expression and HA production in vivo. These data define a novel role for 1,25D3 and the VDR in control of HA synthesis in epithelial tissues that likely contributes to its anti-cancer actions.

TGF-β1 is an epithelial-mesenchymal transition (EMT)-inducing factor that is critical in tumor progression. However, whether the effect of TGF-β1 on breast cancer is through the EMT pathway remains to be determined, and drug development based on this mechanism needs to be improved. Results of this study showed that TGF-β1 dysregulation significantly correlated with the expression levels of EMT-associated markers and transcriptional factors. Exogenous expression of TGF-β1 promoted breast cancer cell metastasis and EMT progression. In addition, direct binding of baicalin to TGF-β1 caused its inactivation, which subsequently blocked signal transduction and inhibited breast cancer cell metastasis. In vivo experiment results further invalidated the inhibitory effect of baicalin on TGF-β1-induced tumor metastasis. These results suggest that baicalin, an active ingredient used in traditional Chinese medicine, exhibits a potential therapeutic effect on breast cancer metastasis by regulating TGF-β1-dependent EMT progression.

The TMEM165 gene encodes for a multiple pass membrane protein localized in the Golgi that has been linked to congenital disorders of glycosylation. The TMEM165 protein is a putative ion transporter that regulates H⁺/Ca⁺⁺/Mn⁺⁺ homeostasis and pH in the Golgi. Previously, we identified TMEM165 as a potential biomarker for breast carcinoma in a glycoproteomic study using late stage invasive ductal carcinoma tissues with patient- matched adjacent normal tissues. The TMEM165 protein was not detected in non-malignant matched breast tissues and was detected in invasive ductal breast carcinoma tissues by mass spectrometry. Our hypothesis is that the TMEM165 protein confers a growth advantage to breast cancer. In this preliminary study we have investigated the expression of TMEM165 in earlier stage invasive ductal carcinoma and ductal carcinoma in situ cases. We created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration, tumor growth, and tumor vascularization in vivo. Furthermore, we find that TMEM165 expression alters the glycosylation of breast cancer cells and these changes promote the invasion and growth of breast cancer by altering the expression levels of key glycoproteins involved in regulation of the epithelial to mesenchymal transition such as E-cadherin. These studies illustrate new potential functions for this Golgi membrane protein in the control of breast cancer growth and invasion.

The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive in vivo. In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels.

Altered cell metabolism is a hallmark of cancer and critical for its development. Particularly, activation of one-carbon metabolism in tumor cells can sustain oncogenesis while contributing to epigenetic changes and metabolic adaptation during tumor progression. We assessed whether increased one-carbon metabolism activity is a metabolic feature of invasive ductal carcinoma (IDC). Differences in the metabolic profile between biopsies from IDC (n = 47) and its adjacent tissue (n = 43) and between biopsies from different breast cancer subtypes were assessed by gas spectrometry in targeted (Biocrates Life Science^®) and untargeted approaches, respectively. The metabolomics data were statistically treated using MetaboAnalyst 4.0, SIMCA P+ (version 12.01), Statistica 10 software and t test with p < 0.05. The Cancer Genome Atlas breast cancer dataset was also assessed to validate the metabolomic profile of IDC. Our targeted metabolomics analysis showed distinct metabolomics profiles between IDC and adjacent tissue, where IDC displayed a comparative enrichment of metabolites involved in one-carbon metabolism (serine, glycine, threonine, and methionine) and a predicted increase in the activity of pathways that receive and donate carbon units (i.e., folate, methionine, and homocysteine). In addition, the targeted and untargeted metabolomics analyses showed similar metabolomics profiles between breast cancer subtypes. The gene set enrichment analysis identified different transcription-related functions between IDC and non-tumor tissues that involved one-carbon metabolism. Our data suggest that one-carbon metabolism may be a central pathway in IDC and even in general breast tumors, representing a potential target for its treatment and prevention.

Clinical, epidemiological and experimental data identified the insulin-like growth factor-1 receptor (IGF1R) as a candidate therapeutic target in oncology. While this paradigm is based on well-established biological facts, including the potent anti-apoptotic and cell survival capabilities of the receptor, most Phase III clinical trials designed to target the IGF1R led to disappointing results. The present study was aimed at evaluating the hypothesis that combined treatment composed of selective IGF1R inhibitor along with classical chemotherapy might be more effective than individual monotherapies in breast cancer treatment. Analyses included comprehensive measurements of the synergism achieved by various combination regimens using the CompuSyn software. In addition, proteomic analyses were conducted to identify the proteins involved in the synergistic killing effect at a global level. Data presented here demonstrates that co-treatment of IGF1R inhibitor along with chemotherapeutic drugs markedly improves the therapeutic efficiency in breast cancer cells. Of clinical relevance, our analyses indicate that high IGF1R baseline expression may serve as a predictive biomarker for IGF1R targeted therapy. In addition, we identified a ten-genes signature with potential predictive value. In conclusion, the use of a series of bioinformatics tools shed light on some of the biological pathways that might be responsible for synergysm in cancer therapy.

Background: Brain metastasis (BM) is an increasingly common and devastating complication of breast cancer (BC).

Methods: A systematic literature search of EMBASE and MEDLINE was conducted to elucidate the current state of knowledge on known and novel prognostic factors associated with 1) the risk for BCBM and 2) the time to brain metastases (TTBM).

Results: A total of 96 studies involving institutional records from 28 countries were identified. Of these, 69 studies reported risk factors of BCBM, 46 factors associated with the TTBM and twenty studies examined variables for both outcomes. Young age, estrogen receptor negativity (ER-), overexpression of human epidermal factor (HER2+), and higher presenting stage, histological grade, tumor size, Ki67 labeling index and nodal involvement were consistently found to be independent risk factors of BCBM. Of these, triple-negative BC (TNBC) subtype, ER-, higher presenting histological grade, tumor size, and nodal involvement were also reported to associate with shorter TTBM. In contrast, young age, hormone receptor negative (HR-) status, higher presenting stage, nodal involvement and development of liver metastasis were the most important risk factors for BM in HER2-positive patients.

Conclusions: The study provides a comprehensive and individual evaluation of the risk factors that could support the design of screening tools and interventional trials for early detection of BCBM.

Exercise is associated with favorable changes in circulating immune cells and improved survival in early-stage breast cancer patients, but the mechansims remain to be fully elucidated. Preclinical studies indicate that physical activity started before tumor injection reduces tumor incidence and progression. Here we tested whether exercise has anti-tumor effects in mice with established 4T1 mammary carcinoma, a mouse model of triple negative breast cancer. Exercise slowed tumor progression and reduced the tumor-induced accumulation of myeloid-derived suppressor cells (MDSCs). The reduction in MDSCs was accompanied by a relative increase in natural killer and CD8 T cell activation, suggesting that exercise restores a favorable immune environment. Consistently, exercise improved responses to a combination of programmed cell death protein 1 (PD-1) blockade and focal radiotherapy. These data support further investigations of exercise in breast cancer patients treated with combinations of immunotherapy and cytotoxic agents to improve cancer outcomes.

Cancers often overexpress anti-apoptotic Bcl-2 proteins for cell death evasion, a recognized hallmark of cancer progression. While estrogen receptor (ER)-α+ breast cancers express high levels of three anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1), pharmacological inhibition of Bcl-2 and/or Bcl-xL fails to induce cell death in ERα+ breast cancer cell lines, due to rapid and robust Mcl-1 upregulation. The mechanisms of acute Mcl-1 upregulation in response to Bcl-2/Bcl-xL inhibition remain undefined in in ERα+ breast cancers. We report here that blockade of Bcl-2 or Bcl-xL, alone or together, rapidly induced mTOR signaling in ERα+ breast cancer cells, rapidly increasing cap-dependent Mcl-1 translation. Cells treated with a pharmacological inhibitor of cap-dependent translation, or with the mTORC1 inhibitor RAD001/everolimus, displayed reduced protein levels of Mcl-1 under basal conditions, and failed to upregulate Mcl-1 protein expression following treatment with ABT-263, a pharmacological inhibitor of Bcl-2 and Bcl-xL. Although treatment with ABT-263 alone did not sustain apoptosis in tumor cells in culture or in vivo, ABT-263 plus RAD001 increased apoptosis to a greater extent than either agent used alone. Similarly, combined use of the selective Mcl-1 inhibitor VU661013 with ABT-263 resulted in tumor cell apoptosis and diminished tumor growth in vivo. These findings suggest that rapid Mcl-1 translation drives ABT-263 resistance, but can be combated directly using emerging Mcl-1 inhibitors, or indirectly through existing and approved mTOR inhibitors.