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Background: We have shown previously that Notch-1 is critical for the development and maintenance of trasuzumab or lapatinib resistance in HER2 positive breast cancer cell lines. Here we sought to identify the critical genes that are regulated by Notch to promote anti-HER2 targeted resistance. Methods: We measured expression of 84 known breast cancer-associated transcripts using a real-time PCR array in both trastuzumab sensitive and resistant BT474 cell lines. A 4 fold increase or decrease in transcripts levels were measured to be statisitically significant. To confirm the change in levels, we designed primers to each gene of interest and repeated the PCR at least three independent times. Furthermore, we specifically asked whether Notch-1 or it's transcriptional mediator, CBF-1, directly regulated any of the identified genes in HER2 positive breast cancer cells using a genetic knocked down approach. An ANOVA for multiple comparisons was used to compute statistical significance. Results: We identified more than 4 fold increase in APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts in trastuzumab resistant versus sensitive cells. Conversley, Gli1 was downregulated in resistant versus sensitive cells. Interestingly, siRNA directed against Notch-1 or CBF-1 decreased ABCG2 and Gata-3, but conversely increased ABCB1 and HRG-2 in resistant cells. Conclusions: Our findings demonstrate APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts are significantly increased in trastuzumab resistant breast cancer cells compared to sensitive cells. More importantly, Notch-1 or it's direct transcriptional activator CBF-1 may contribute to traztuzumab resistance by directly regulating critical genes that contribute to multidrug resistance: ABCB1 and ABCG2 and/or genes associated with stem cell survival: Gata-3 and HRG-2. [ABSTRACT FROM AUTHOR]

Background: Treatment options for patients presenting with TNBC are limited, primarily because there is no approved targeted therapy available. Canonical breast cancer targets such as estrogen receptor and HER2/neu proteins are absent in TNBC. Furthermore, there is an urgent need to uncover biomarkers in this disease in light of its costly treatment as compared to non-TNBC treatment and, more importantly, its poor prognosis as evidenced by aggressive tumor metastasis and high patient mortality. While Notch-1 target gene expression has been reported to be linked to various types of malignancies, we propose that since Notch signaling pathway is involved in cancer cell proliferation and survival it could be a likely oncogenic driver and possible target in certain TNBC patients. Methods: Human TNBC cell lines BT-549, MDA-MB-231, and MDA-MB-468 were used to measure endogenous relative mRNA transcript levels of Notch genes and gene targets by means of real time PCR. In addition, the RNA from formalin-fixed, paraffin-embedded specimen from women-diagnosed with TNBC who had undergone breast surgery was also analyzed for comparable gene expression for similar targets following Laser capture micro- dissection. Results: The in vitro results show that mRNA transcripts of Notch-4, Deltex-1, Hes-1 and - Hes5, as well as upstream Notch regulator PEA3 targets IL-8 and MMP-9 were increased in MDA-MB-468 and BT-549 cells as compared to MDA-MB-231 cells (n = 3-5). In the clinical sample specimens, Notch-1, Jagged-1, and Hes-5 genes were up-regulated, while the Notch-4 gene was down-regulated in TNBC patients as compared to normal control specimens from reduction mammoplasty (n = 4). Conclusions: Our data present the heterogeneity of TNBC disease. They also indicate that the Notch signaling pathway is up-regulated in certain TNBC cell lines and human TNBC breast cancer tissue. Lastly, these results suggest that some components of Notch signaling may be viable biomarkers to better predict Notch activity for future therapeutic interventions using Notch inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: Many well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis -derived EPS affects cancer., Methods: This work investigated effects of EPS on phenotypes of breast cancer cells as a cancer model. The phenotypes included proliferation, mammosphere formation, cell migration, and tumor growth in two immune compromised mouse models. RNA sequencing was performed on RNA from four breast cancer cells treated with PBS or EPS. IKKβ or STAT1 signaling was assessed using pharmacologic or RNAi-mediated knock down approaches., Results: Short-term treatment with EPS inhibited proliferation of certain breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-κB. IKKβ and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells., Discussion: These results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer cell phenotypes. These results warrant future studies in immune competent mice and different cancer models to fully understand potential benefits and/or side effects of long-term use of probiotics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Nguyen, Ma, Wyatt, Knight and Osipo.)

Breast cancer therapies have made significant strides in improving survival for patients over the past decades. However, recurrence and drug resistance continue to challenge long-term recurrence-free and overall survival rates. Mounting evidence supports the cancer stem cell model in which the existence of a small population of breast cancer stem cells (BCSCs) within the tumor enables these cells to evade conventional therapies and repopulate the tumor, giving rise to more aggressive, recurrent tumors. Thus, successful breast cancer therapy would need to target these BCSCs, as well the tumor bulk cells. Since the Women's Health Initiative study reported an increased risk of breast cancer with the use of conventional hormone replacement therapy in postmenopausal women, many have turned their attention to phytoestrogens as a natural alternative. Phytoestrogens are plant compounds that share structural similarities with human estrogens and can bind to the estrogen receptors to alter the endocrine responses. Recent studies have found that phytoestrogens can also target BCSCs and have the potential to complement conventional therapy eradicating BCSCs. This review summarized the latest findings of different phytoestrogens and their effect on BCSCs, along with their mechanisms of action, including selective estrogen receptor binding and inhibition of molecular pathways used by BCSCs. The latest results of phytoestrogens in clinical trials are also discussed to further evaluate the use of phytoestrogen in the treatment and prevention of breast cancer.

Breast cancer is a heterogeneous disease that can be subdivided into unique molecular subtypes based on protein expression of the Estrogen Receptor, Progesterone Receptor, and/or the Human Epidermal Growth Factor Receptor 2. Therapeutic approaches are designed to inhibit these overexpressed receptors either by endocrine therapy, targeted therapies, or combinations with cytotoxic chemotherapy. However, a significant percentage of breast cancers are inherently resistant or acquire resistance to therapies, and mechanisms that promote resistance remain poorly understood. Notch signaling is an evolutionarily conserved signaling pathway that regulates cell fate, including survival and self-renewal of stem cells, proliferation, or differentiation. Deregulation of Notch signaling promotes resistance to targeted or cytotoxic therapies by enriching of a small population of resistant cells, referred to as breast cancer stem cells, within the bulk tumor; enhancing stem-like features during the process of de-differentiation of tumor cells; or promoting epithelial to mesenchymal transition. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance through reduction or elimination of breast cancer stem cells. However, Notch inhibitors have yet to be clinically approved for the treatment of breast cancer, mainly due to dose-limiting gastrointestinal toxicity. In this review, we discuss potential mechanisms of Notch-mediated resistance in breast cancer cells and breast cancer stem cells, and various methods of targeting Notch through γ-secretase inhibitors, Notch signaling biologics, or transcriptional inhibitors. We also discuss future plans for identification of novel Notch-targeted therapies, in order to reduce toxicity and improve outcomes for women with resistant breast cancer.

Recurrence of estrogen receptor (ER)-positive breast tumors despite curative-intent adjuvant therapy is thought to be due to enrichment of tumor initiating cells (TIC) during endocrine therapy (ET). Recently, it was identified that by antagonizing the ER, ET promotes rapid degradation of the death-associated factor 6 (DAXX) protein, which is necessary and sufficient to potently inhibit TICs. Thus, the goal of the current study was to identify a DAXX-inducing agent to inhibit TICs and prevent proliferation of the tumor. Phytoestrogens (naringenin, resveratrol, genistein, apigenin, and quercetin) were screened for DAXX protein expression, anti-TIC and anti-proliferative efficacy in vitro and in vivo. Specific DAXX-inducing phytoestrogens were tested to assess selectivity towards ERα and/or ERβ. Results showed that phytoestrogens tested induced DAXX protein expression and inhibited survival of TICs from ER+ MCF-7 and T47D cells. Only naringenin, resveratrol, and quercetin did not stimulate total cell proliferation. Naringenin, resveratrol, but not quercetin inhibited survival of TICs in vitro and in vivo in a DAXX-dependent manner. Naringenin-induced DAXX protein expression and inhibition of TICs seemed to be more selective towards ERβ while resveratrol was more selective through ERα. Naringenin or resveratrol inhibited the rate of tumor initiation and rate of tumor growth in a DAXX-dependent manner. These results suggest that a therapeutic approach using a phytoestrogen to induce DAXX protein expression could potently inhibit TICs within a tumor to delay or prevent tumor initiation. Therefore, a DAXX-promoting phytoestrogen should be explored for prevention of tumor progression in advanced disease and relapse in the adjuvant setting., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Estrogen receptor (ER)-positive breast cancer recurrence is thought to be driven by tumor-initiating cells (TIC). TICs are enriched by endocrine therapy through NOTCH signaling. Side effects have limited clinical trial testing of NOTCH-targeted therapies. Death-associated factor 6 (DAXX) is a newly identified marker whose RNA expression inversely correlates with NOTCH in human ER + breast tumor samples. In this study, knockdown and overexpression approaches were used to investigate the role of DAXX on stem/pluripotent gene expression, TIC survival in vitro , and TIC frequency in vivo , and the mechanism by which DAXX suppresses TICs in ER + breast cancer. 17β-Estradiol (E 2 )-mediated ER activation stabilized the DAXX protein, which was required for repressing stem/pluripotent genes ( NOTCH4, SOX2, OCT4, NANOG , and ALDH1A1 ), and TICs in vitro and in vivo . Conversely, endocrine therapy promoted rapid protein depletion due to increased proteasome activity. DAXX was enriched at promoters of stem/pluripotent genes, which was lost with endocrine therapy. Ectopic expression of DAXX decreased stem/pluripotent gene transcripts to levels similar to E 2 treatment. DAXX-mediated repression of stem/pluripotent genes and suppression of TICs was dependent on DNMT1. DAXX or DNMT1 was necessary to inhibit methylation of CpGs within the SOX2 promoter and moderately within the gene body of NOTCH4, NOTCH activation, and TIC survival. E 2 -mediated stabilization of DAXX was necessary and sufficient to repress stem/pluripotent genes by recruiting DNMT1 to methylate some promoters and suppress TICs. These findings suggest that a combination of endocrine therapy and DAXX-stabilizing agents may inhibit ER + tumor recurrence. SIGNIFICANCE: Estradiol-mediated stabilization of DAXX is necessary and sufficient to repress genes associated with stemness, suggesting that the combination of endocrine therapy and DAXX-stabilizing agents may inhibit tumor recurrence in ER + breast cancer., (©2019 American Association for Cancer Research.)

Purpose: HER2-positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as cancer stem cells (CSC). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. NOTCH promotes breast CSC survival and self-renewal, and overexpression of NOTCH1 and the NOTCH ligand JAGGED1 predict poor outcome. Resistance to anti-HER2 therapy in HER2 + breast cancer requires NOTCH1, and that combination of trastuzumab and a gamma secretase inhibitor (GSI) prevents tumor relapse in xenograft models. Experimental Design: The current study investigates mechanisms by which HER2 tyrosine kinase activity regulates NOTCH-dependent CSC survival and tumor initiation. Results: Lapatinib-mediated HER2 inhibition shifts the population of HER2 + breast cancer cells from low membrane JAGGED1 expression to higher levels, independent of sensitivity to anti-HER2 treatment within the bulk cell population. This increase in membrane JAGGED1 is associated with higher NOTCH receptor expression, activation, and enrichment of CSCs in vitro and in vivo Importantly, lapatinib treatment results in growth arrest and cell death of JAGGED1 low-expressing cells while the JAGGED1 high-expressing cells continue to cycle. High membrane JAGGED1 protein expression predicts poor overall cumulative survival in women with HER2 + breast cancer. Conclusions: These results indicate that higher membrane JAGGED1 expression may be used to either predict response to anti-HER2 therapy or for detection of NOTCH-sensitive CSCs posttherapy. Sequential blockade of HER2 followed by JAGGED1 or NOTCH could be more effective than simultaneous blockade to prevent drug resistance and tumor progression. Clin Cancer Res; 24(18); 4566-78. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

Potassium ion (K + ) channels have been recently found to play a critical role in cancer biology. Despite that pharmacologic manipulation of ion channels is recognized as an important therapeutic approach, very little is known about the effects of targeting of K + channels in cancer. In this study, we demonstrate that use of the Kv11.1 K + channel activator NS1643 inhibits tumor growth in an in vivo model of breast cancer. Tumors exposed to NS1643 had reduced levels of proliferation markers, high expression levels of senescence markers, increased production of ROS and DNA damage compared to tumors of untreated mice. Importantly, mice treated with NS1643 did not exhibit significant cardiac dysfunction. In conclusion, pharmacological stimulation of Kv11.1 activity produced arrested TNBC-derived tumor growth by generating DNA damage and senescence without significant side effects. We propose that use of Kv11.1 channels activators could be considered as a possible pharmacological strategy against breast tumors., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Deubiquitinases (DUB) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressor signaling. Here, evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, because OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared with normal samples. The global OTUD6B expression level does not change significantly between nonneoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch., Implications: Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Mol Cancer Res; 15(2); 117-27. ©2016 AACR., (©2016 American Association for Cancer Research.)

Human Epidermal Growth Factor Receptor type 2 (HER2) gene amplification and/or protein overexpression is observed in patients suffering from HER2+ breast cancer. This subtype of breast cancer has improved prognosis due to availability of anti-HER2 therapy. However, drug resistance and tumor recurrence still remains a major concern. Cancer Stem Cells (CSCs) are believed to constitute the subset of cell population that is resistant to drug treatment and possesses characteristics of stem cells. CSCs enable the tumors to thrive despite major insults. This review provides a comprehensive idea about the concept of CSCs in context of HER2+ breast cancer by providing the description of the markers that are used for the identification of CSCs and by elucidating the signaling pathways that are associated with HER2+ breast CSCs. Furthermore, the review also describes the interaction of HER2 with those signaling pathways and the future of targeting CSCs in HER2+ breast cancer., Competing Interests: Conflicts of interest The authors declare no conflict of interest.

Acquired or de novo resistance to trastuzumab remains a barrier to patient survival and mechanisms underlying this still remain unclear. Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare proteome profiles between trastuzumab sensitive/resistant cells, we identified autophagy related protein 9A (ATG9A) as a down-regulated protein in trastuzumab resistant cells (BT474-TR). Interestingly, ATG9A ectopic expression markedly decreased the proliferative ability of BT474-TR cells but not that of the parental line (BT474). This was accompanied by a reduction of Her2 protein levels and AKT phosphorylation (S473), as well as a decrease in Her2 stability, which was also observed in JIMT1 and MDA-453, naturally trastuzumab-resistant cells. In addition, ATG9A indirectly promoted c-Cbl recruitment to Her2 on T1112, a known c-Cbl docking site, leading to increased K63 Her2 polyubiquitination. Whereas silencing c-Cbl abrogated ATG9A repressive effects on Her2 and downstream PI3K/AKT signaling, its depletion restored BT474-TR proliferative rate. Taken together, our findings show for this first time that ATG9A loss in trastuzumab resistant cells allowed Her2 to escape from lysosomal targeted degradation through K63 poly-ubiquitination via c-Cbl. This study identifies ATG9A as a potentially druggable target to overcome resistance to anti-Her2 blockade., Competing Interests: The authors declare no competing financial interests.

Purpose: Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2-positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2-positive breast cancer., Experimental Design: Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib., Results: The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2 (i) restricts the availability of Jagged-1 at the cell surface to transactivate Notch, (ii) restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation, (iii) reverses trastuzumab resistance in vivo, and (iv) predicts better outcome in women with ErbB-2-positive breast cancer., Conclusions: The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2-positive breast cancer. Moreover, women with ErbB-2-positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy., (©2015 American Association for Cancer Research.)

BRCA1 is an important player in the DNA damage response signaling, and its deficiency results in genomic instability. A complete loss or significantly reduced BRCA1 protein expression is often found in sporadic breast cancer cases despite the absence of genetic or epigenetic aberrations, suggesting the existence of other regulatory mechanisms controlling BRCA1 protein expression. Herein, we demonstrate that Fyn-related kinase (Frk)/Rak plays an important role in maintaining genomic stability, possibly in part through positively regulating BRCA1 protein stability and function via tyrosine phosphorylation on BRCA1 Tyr1552. In addition, Rak deficiency confers cellular sensitivity to DNA damaging agents and poly(ADP-ribose) polymerase (PARP) inhibitors. Overall, our findings highlight a critical role of Rak in the maintenance of genomic stability, at least in part, through protecting BRCA1 and provide novel treatment strategies for patients with breast tumors lacking Rak., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflict of interest.

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼ 15-20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2-) breast cancer tissues, whereas both HER2+ and HER2- tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser(674) by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Notch pathway is a well-established mediator of cell-cell communication that plays a critical role in stem cell survival, self-renewal, cell fate decisions, tumorigenesis, invasion, metastasis, and drug resistance in a variety of cancers. An interesting form of crosstalk exists between the Notch receptor and the Epidermal Growth Factor Receptor Tyrosine Kinase family, which consists of HER-1, -2, -3, and -4. Overexpression of HER and/or Notch occurs in several human cancers including brain, lung, breast, ovary, and skin making them potent oncogenes capable of advancing malignant disease. Continued assessment of interplay between these two critical signaling networks uncovers new insight into mechanisms used by HER-driven cancer cells to exploit Notch as a compensatory pathway. The compensatory Notch pathway maintains HER-induced downstream signals transmitted to pathways such as Mitogen Activated Protein Kinase and Phosphatidylinositol 3-Kinase (PI3K), thereby allowing cancer cells to survive molecular targeted therapies, undergo epithelial to mesenchymal transitioning, and increase cellular invasion. Uncovering the critical crosstalk between the HER and Notch pathways can lead to improved screening for the expression of these oncogenes enabling patients to optimize their personal treatment options and predict potential treatment resistance. This review will focus on the current state of crosstalk between the HER and Notch receptors and the effectiveness of current therapies targeting HER-driven cancers.

Background: The purpose of this study was to evaluate whether breast carcinoma biomarkers vary among separate tumor foci of multifocal/multicentric (MF/MC) breast carcinomas and whether this variation correlates with morphological features and tumor grade., Design: We reviewed the biomarker profiles of MF/MC invasive breast carcinomas diagnosed between January 2001 and June 2010 at our institution. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal receptor protein (HER2) results were classified as positive or negative., Results: Out of the 51 patients included in the study, 6 cases had 2 tumor foci with different morphologies, 7 cases had 2 foci with similar morphology but different grades, and 38 cases had 2 tumor foci with similar morphologies and grades. Out of the 38 patients who had MF/MC tumors with the same morphology and grade, only 1 patient had a difference in ER and PR status between foci. Out of the 7 patients who had morphologically similar tumors with different grades, 4 had similar results in both tumor foci, 3 had different results for ER and PR, and another had differing results for HER2 between the foci. All 6 patients who had MF/MC foci with different morphologies exhibited similar ER, PR, and HER2 results between the foci., Conclusion: Regardless of the similarity in tumor morphology or grade, a small number of cases included foci that exhibited different tumor marker expression, which might affect the treatment strategy. Therefore, our results suggest that the evaluation of tumor markers in different foci should be considered in MF/MC tumors for accurate treatment strategies.

The term "triple-negative breast cancer" (TNBC) is a heterogeneous subtype of breast cancer. Unfortunately, due to the lack of expression of hormone receptors and human epidermal growth factor receptor-2, therefore the lack of US Food and Drug Administration-approved targeted therapies, TNBC has the worst prognosis of all subtypes of breast cancer. Notch signaling has emerged as a pro-oncogene in several human malignancies and has particularly been associated with the triple-negative subtype of breast cancer. This chapter explores the role of Notch signaling in triple negative and other subtypes of breast cancer, the relationship of Notch with other breast cancer biomarkers, prognostic indicators associated with Notch, and potential therapeutic strategies targeting Notch inhibition. Hopefully, better understanding of this signaling pathway in the future will lead to optimal molecular therapeutic treatments for TNBC patients, improving their quality of life and outcome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims. To compare the results of estrogen and progesterone receptors (ER, PR), and human epidermal growth factor receptor-2 (HER2) expression status on biopsy and excision specimens and to evaluate the effect of cold ischemia time and/or formalin fixation on these biomarkers. Methods. Breast carcinomas that were diagnosed between 2007 and 2009 by core needle biopsy, and subsequently excised in our institution, were included in the study. Data regarding the tumor morphology, grade, and ER, PR, and HER2 status were retrospectively collected from the pathology reports. Results. Five out of 149 (3.4%) cases with ER-positive receptor status in the biopsy specimen became ER-negative in the subsequent excision specimen. Nine out of 126 (7.1%) cases with PR-positive receptor status in the biopsy specimen became PR-negative in the excision specimen. Receptor status change was predominantly seen in tumors with low ER and PR receptor expression. HER2 results were consistent between biopsy and excision specimens in all cases tested. Conclusions. Cold ischemia time and/or formalin fixation affect mainly ER and PR testing with low Allred scores and support the implementation of the ASCO/CAP guidelines. HER2 results, however, were not affected in our limited number of patients.

Introduction: Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells., Methods: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both., Results: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo., Conclusions: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.

The cellular heterogeneity of neoplasms has been at the center of considerable interest since the "cancer stem cell hypothesis", originally formulated for hematologic malignancies, was extended to solid tumors. The origins of cancer "stem" cells (CSC) or tumor-initiating cells (TIC; henceforth referred to as CSCs) and the methods to identify them are hotly debated topics. Nevertheless, the existence of subpopulations of tumor cells with stem-like characteristics has significant therapeutic implications. The stem-like phenotype includes indefinite self-replication, pluripotency, and, importantly, resistance to chemotherapeutics. Thus, it is plausible that CSCs, regardless of their origin, may escape standard therapies and cause disease recurrences and/or metastasis after apparently complete remissions. Consequently, the idea of selectively targeting CSCs with novel therapeutics is gaining considerable interest. The Notch pathway is one of the most intensively studied putative therapeutic targets in CSC, and several investigational Notch inhibitors are being developed. However, successful targeting of Notch signaling in CSC will require a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to prove safe and effective. Additionally, to determine which patients are most likely to benefit from treatment with Notch-targeting therapeutics, reliable biomarkers to measure pathway activity in CSC from specific tumors will have to be identified and validated. This article summarizes the most recent developments in the field of Notch-targeted cancer therapeutics, with emphasis on CSC., ((c) 2010 AACR.)

Epidermal growth factor receptor-2 (ErbB-2/HER2) is a potent breast oncogene that has been shown to be amplified in 20% of breast cancers. Overexpression of ErbB-2 predicts for aggressive tumor behavior, resistance to some cytotoxic and antihormonal therapies, and poor overall survival. Trastuzumab, the humanized, monoclonal antibody directed against ErbB-2 has shown tremendous efficacy and improved overall survival for women when combined with a taxane-based chemotherapy. However, resistance to trastuzumab remains a major concern, most notably in women with metastatic breast cancer. Numerous mechanisms that include overexpression of alternate receptor tyrosine kinases and/or loss of critical tumor suppressors have been proposed in the last several years to elucidate trastuzumab resistance. Here we review the many possible mechanisms of action that could contribute to resistance, and novel therapies to prevent or reverse the resistant phenotype. Moreover, we provide a critical role for Notch signaling cross-talk with overlapping or new signaling networks in trastuzumab-resistant breast.