The Effect of Cold Ischemia Time and/or Formalin Fixation on Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor-2 Results in Breast Carcinoma

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

NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications

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 G(1 )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

Notch versus the proteasome: what is the target of γ-secretase inhibitor-I?

γ-Secretase inhibitors are new anti-cancer agents targeting Notch signaling. Their specificity for Notch is as yet unclear. Han and colleagues investigated the effects of Z-LeuLeuNleu-CHO on growth of breast cancer cells. The results demonstrated a reduction in cell viability primarily via proteasome inhibition independent of Notch activity. Currently, γ-secretase inhibitors in clinical trials are structurally distinct from Z-LeuLeuNleu-CHO. Their effects on the proteasome are yet to be determined. However, findings from Han and colleagues pose two critical questions: Is the level of proteasomal activity in breast tumors the driving force for growth? What does the Notch pathway contribute to this growth

ATG9A loss confers resistance to trastuzumab via c-Cbl mediated Her2 degradation

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

The effect of an exopolysaccharide probiotic molecule from Bacillus subtilis on breast cancer cells

IntroductionMany 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.MethodsThis 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. ResultsShort-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. DiscussionThese 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

Clinical relevance of "withdrawal therapy" as a form of hormonal manipulation for breast cancer

<p>Abstract</p> <p>Background</p> <p>It has been shown in in-vitro experiments that "withdrawal" of tamoxifen inhibits growth of tumor cells. However, evidence is scarce when this is extrapolated into clinical context. We report our experience to verify the clinical relevance of "withdrawal therapy".</p> <p>Methods</p> <p>Breast cancer patients since 1998 who fulfilled the following criteria were selected from the departmental database and the case-notes were retrospectively reviewed: (1) estrogen receptor positive, operable primary breast cancer in elderly (age > 70 years), locally advanced or metastatic breast cancer; (2) disease deemed suitable for treatment by hormonal manipulation; (3) disease assessable by UICC criteria; (4) received "withdrawal" from a prior endocrine agent as a form of therapy; (5) on "withdrawal therapy" for ≥ 6 months unless they progressed prior.</p> <p>Results</p> <p>Seventeen patients with median age of 84.3 (53.7-92.5) had "withdrawal therapy" as second to tenth line of treatment following prior endocrine therapy using tamoxifen (n = 10), an aromatase inhibitor (n = 5), megestrol acetate (n = 1) or fulvestrant (n = 1). Ten patients (58.8%) had clinical benefit (CB) (complete response/partial response/stable disease ≥ 6 months) with a median duration of Clinical Benefit (DoCB) of 10+ (7-27) months. Two patients remain on "withdrawal therapy" at the time of analysis.</p> <p>Conclusion</p> <p>"Withdrawal therapy" appears to produce sustained CB in a significant proportion of patients. This applies not only to "withdrawal" from tamoxifen, but also from other categories of endocrine agents. "Withdrawal" from endocrine therapy is, therefore, a viable intercalating option between endocrine agents to minimise resistance and provide additional line of therapy. It should be considered as part of the sequencing of endocrine therapy.</p

Efficacy and tolerability of high dose "ethinylestradiol" in post-menopausal advanced breast cancer patients heavily pre-treated with endocrine agents

BACKGROUND: High dose estrogens (HDEs) were frequently used as endocrine agents prior to the introduction of tamoxifen which carries fewer side effects. Due to the development of resistance to available endocrine agents in almost all women with metastatic breast cancer, interest has renewed in the use of HDEs as yet another endocrine option that may have activity. We report our experience with one of the HDEs ("ethinylestradiol" 1 mg daily) in advanced breast cancer (locally advanced and metastatic) in post-menopausal women who had progressed on multiple endocrine agents. PATIENTS AND METHODS: According to a database of advanced breast cancer patients seen in our Unit since 1998, those who had complete set of information and fulfilled the following criteria were studied: (1) patients in whom further endocrine therapy was deemed appropriate i.e., patients who have had clinical benefit with previous endocrine agents or were not fit or unwilling to receive chemotherapy in the presence of potentially life-threatening visceral metastases; (2) disease was assessable by UICC criteria; (3) were treated with "ethinylestradiol" until they were withdrawn from treatment due to adverse events or disease progression. RESULTS: Twelve patients with a median age of 75.1 years (49.1 – 85 years) were identified. Majority (N = 8) had bony disease. They had ethinylestradiol as 3(rd )to 7(th )line endocrine therapy. One patient (8%) came off treatment early due to hepato-renal syndrome. Clinical benefit (objective response or durable stable disease for ≥ 6 months) was seen in 4 patients (33.3%) with a median duration of response of 10+ (7–36) months. The time to treatment failure was 4 (0.5–36) months. CONCLUSION: Yet unreported, high dose "ethinylestradiol" is another viable therapeutic strategy in heavily pre-treated patients when further endocrine therapy is deemed appropriate. Although it tends to carry more side effects, they may not be comparable to those of other HDEs (such as diethylstilbestrol) or chemotherapy

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival

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

Inhibition of HER2 enriches for Jagged1-dependent breast cancer stem cells: role for membrane Jagged1

Purpose: Human Epidermal Growth Factor Receptor 2 (HER2) positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as Cancer Stem Cells (CSCs). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. Notch promotes breast CSCs 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 expressing 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 post therapy. Sequential blockade of HER2 followed by Jagged1 or Notch could be more effective than simultaneous blockade to prevent drug resistance and tumor progression