Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells

The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 mumol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor-negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor-negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy.

Switching of vascular phenotypes within a murine breast cancer model induced by angiopoietin-2

Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Sentinel lymph node biopsy is associated with improved survival compared to level I & II axillary lymph node dissection in node negative breast cancer patients

OBJECTIVE: The few long-term follow-up data for sentinel lymph node (SLN) negative breast cancer patients demonstrate a 5-year disease-free survival of 96-98%. It remains to be elucidated whether the more accurate SLN staging defines a more selective node negative patient group and whether this is associated with better overall and disease-free survival compared with level I ; II axillary lymph node dissection (ALND). METHODS: Three-hundred and fifty-five consecutive node negative patients with early stage breast cancer (pT1 and pT2< or =3 cm, pN0/pN(SN)0) were assessed from our prospective database. Patients underwent either ALND (n=178) in 1990-1997 or SLN biopsy (n=177) in 1998-2004. All SLN were examined by step sectioning, stained with H;E and immunohistochemistry. Lymph nodes from ALND specimens were examined by standard H;E only. Neither immunohistochemistry nor step sections were performed in the analysis of ALND specimen. RESULTS: The median follow-up was 49 months in the SLN and 133 months in the ALND group. Patients in the SLN group had a significantly better disease-free (p=0.008) and overall survival (p=0.034). After adjusting for other prognostic factors in Cox proportional hazard regression analysis, SLN procedure was an independent predictor for improved disease-free (HR: 0.28, 95% CI: 0.10-0.73, p=0.009) and overall survival (HR: 0.34, 95% CI: 0.14-0.84, p=0.019). CONCLUSIONS: This is the first prospective analysis providing evidence that early stage breast cancer patients with a negative SLN have an improved disease-free and overall survival compared with node negative ALND patients. This is most likely due to a more accurate axillary staging in the SLN group.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Does subjective burden of early breast cancer and its treatment affect immune measures during adjuvant therapy?

Psychosocial factors have been described as affecting cellular immune measures in healthy subjects. In patients with early breast cancer we explored bi-directional psycho-immune effects to determine whether subjective burden has an impact on immune measures, and vice versa. Patients (n = 239) operated for early breast cancer and randomized into International Breast Cancer Study Group (IBCSG) adjuvant clinical trials were assessed immediately before the beginning of adjuvant treatment (baseline) and 3 and 6 months thereafter, at the beginning of the corresponding treatment cycle. Cellular immune measures (leukocytes, lymphocytes, lymphocyte subset counts), markers of activation of the cellular immune system (beta2-microglobulin, soluble interleukin-2 receptor serum levels), and self-report subjective burden (global indicators of physical well-being, mood, coping effort) were assessed concurrently. The relationship between subjective burden and gradients of immune measures was investigated with regression analyses controlling for adjuvant treatment. There was a pattern of small negative associations between all variables assessing subjective burden before the beginning of adjuvant therapy with the gradients of the markers of activation of the cellular immune system and NK cell counts. In particular, better mood predicted a decline in the course of beta2-microglobulin and IL-2r at months 3 and 6. The gradient of beta2-microglobulin was associated with mood and coping effort at month 3. However, the effect sizes were very small. In conclusion, in this explorative investigation, there was an indication for subjective burden affecting and being affected by markers of activation of the cellular immune system during the first 3 and 6 months of adjuvant therapy. The question of clinical significance remains unanswered. These associations have to be investigated with refined assessment tools and schedules.

Adjuvant breast cancer treatment and cognitive function: current knowledge and research directions

Evidence is mounting that potentially curative systemic adjuvant therapy for early-stage breast cancer may result in cognitive impairment. Five published studies have investigated cognitive function in this setting, and the consistent results of all five studies suggest an adverse effect of adjuvant chemotherapy. These studies are reviewed with particular attention to their methodologic limitations. For example, all five studies used cross-sectional designs, none controlled for possible confounding hormonal factors, and three examined patients who had not received a uniform chemotherapy regimen. The potential roles of chemotherapy-induced menopause and of adjuvant hormonal therapy in cognitive impairment are also discussed. Priorities for future research include confirmation of an effect of adjuvant chemotherapy in a study with a longitudinal design, closer examination of the potential contribution of hormonal factors, and similar studies on the effect of adjuvant therapy on cognitive function in other cancer types. If an effect of systemic adjuvant therapy on cognitive function is confirmed, such an effect will have implications for informed consent. It may also result in incorporation of objective measures of cognition in clinical trials of adjuvant therapy and in the investigation of preventive interventions that might minimize the impact of cognitive dysfunction after cancer treatment.

Incident malignancies among older long-term breast cancer survivors and an age-matched and site-matched nonbreast cancer comparison group over 10 years of follow-up

BACKGROUND Of the approximately 2.4 million American women with a history of breast cancer, 43% are aged ≥ 65 years and are at risk for developing subsequent malignancies. METHODS Women from 6 geographically diverse sites included 5-year breast cancer survivors (N = 1361) who were diagnosed between 1990 and 1994 at age ≥ 65 years with stage I or II disease and a comparison group of women without breast cancer (N = 1361). Women in the comparison group were age-matched and site-matched to breast cancer survivors on the date of breast cancer diagnosis. Follow-up began 5 years after the index date (survivor diagnosis date or comparison enrollment date) until death, disenrollment, or through 15 years after the index date. Data were collected from medical records and electronic sources (cancer registry, administrative, clinical, National Death Index). Analyses included descriptive statistics, crude incidence rates, and Cox proportional hazards regression models for estimating the risk of incident malignancy and were adjusted for death as a competing risk. RESULTS Survivors and women in the comparison group were similar: >82% were white, 55% had a Charlson Comorbidity Index of 0, and ≥ 73% had a body mass index ≤ 30 kg/m(2) . Of all 306 women (N = 160 in the survivor group, N = 146 in the comparison group) who developed a first incident malignancy during follow-up, the mean time to malignancy was similar (4.37 ± 2.81 years vs 4.03 ± 2.76 years, respectively; P = .28), whereas unadjusted incidence rates were slightly higher in survivors (1882 vs 1620 per 100,000 person years). The adjusted hazard of developing a first incident malignancy was slightly elevated in survivors in relation to women in the comparison group, but it was not statistically significant (hazard ratio, 1.17; 95% confidence interval, 0.94-1.47). CONCLUSIONS Older women who survived 5 years after an early stage breast cancer diagnosis were not at an elevated risk for developing subsequent incident malignancies up to 15 years after their breast cancer diagnosis.

AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therapy

Chemotherapy for isolated locoregional recurrence of breast cancer (CALOR): a randomised trial.

BACKGROUND Patients with isolated locoregional recurrences (ILRR) of breast cancer have a high risk of distant metastasis and death from breast cancer. We aimed to establish whether adjuvant chemotherapy improves the outcome of such patients. METHODS The CALOR trial was a pragmatic, open-label, randomised trial that accrued patients with histologically proven and completely excised ILRR after unilateral breast cancer who had undergone a mastectomy or lumpectomy with clear surgical margins. Eligible patients were enrolled from hospitals worldwide and were centrally randomised (1:1) to chemotherapy (type selected by the investigator; multidrug for at least four courses recommended) or no chemotherapy, using permuted blocks, and stratified by previous chemotherapy, oestrogen-receptor and progesterone-receptor status, and location of ILRR. Patients with oestrogen-receptor-positive ILRR received adjuvant endocrine therapy, radiation therapy was mandated for patients with microscopically involved surgical margins, and anti-HER2 therapy was optional. The primary endpoint was disease-free survival. All analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00074152. FINDINGS From Aug 22, 2003, to Jan 31, 2010, 85 patients were randomly assigned to receive chemotherapy and 77 were assigned to no chemotherapy. At a median follow-up of 4·9 years (IQR 3·6-6 ·0), 24 (28%) patients had disease-free survival events in the chemotherapy group compared with 34 (44%) in the no chemotherapy group. 5-year disease-free survival was 69% (95% CI 56-79) with chemotherapy versus 57% (44-67) without chemotherapy (hazard ratio 0·59 [95% CI 0·35-0·99]; p=0·046). Adjuvant chemotherapy was significantly more effective for women with oestrogen-receptor-negative ILRR (pinteraction=0·046), but analyses of disease-free survival according to the oestrogen-receptor status of the primary tumour were not statistically significant (pinteraction=0·43). Of the 81 patients who received chemotherapy, 12 (15%) had serious adverse events. The most common adverse events were neutropenia, febrile neutropenia, and intestinal infection. INTERPRETATION Adjuvant chemotherapy should be recommended for patients with completely resected ILRR of breast cancer, especially if the recurrence is oestrogen-receptor negative. FUNDING US Department of Health and Human Services, Swiss Group for Clinical Cancer Research (SAKK), Frontier Science and Technology Research Foundation, Australian and New Zealand Breast Cancer Trials Group, Swedish Cancer Society, Oncosuisse, Cancer Association of South Africa, Foundation for Clinical Research of Eastern Switzerland (OSKK), Grupo Español de Investigación en Cáncer de Mama (GEICAM), and the Dutch Breast Cancer Trialists' Group (BOOG).

Accelerated partial breast irradiation with interstitial brachytherapy as second conservative treatment for ipsilateral breast tumour recurrence: multicentric study of the GEC-ESTRO Breast Cancer Working Group

PURPOSE To analyse the clinical outcome after salvage lumpectomy and multi-catheter brachytherapy (MCB) for ipsilateral breast tumour recurrence (IBTR). MATERIAL AND METHODS Between 09/00 and 09/10, 217 patients presenting an IBTR underwent lumpectomy and MCB (low, pulsed, or high-dose rate). Survival rates without second local recurrence (2nd LR), distant metastasis (DM), and overall survival (OS) were analysed as well as late effects and cosmetic results. Univariate and multivariate analyses (MVA) based on IBTR data were performed to find prognostic factors for 2nd LR, DM, and OS. RESULTS Median follow-up after the IBTR was 3.9 years [range: 1.1-10.3]. Five and 10-year actuarial 2nd LR rates were 5.6% [range: 1.5-9.5] and 7.2% [range: 2.1-12.1], respectively. Five and 10-year actuarial DM rates were 9.6% [range: 5.7-15.2] and 19.1% [range: 7.8-28.3], respectively. Five and 10-year actuarial OS rates were 88.7% [range: 83.1-94.8] and 76.4% [range: 66.9-87.3], respectively. In MVA, histological grade was prognostic factor for 2nd LR (p=0.008) and OS (p=0.02); while tumour size was prognostic factor for DM (p=0.03). G3-4 complication rate was 11%. Excellent/good cosmetic result was achieved in 85%. CONCLUSION This study suggests that in case of IBTR, lumpectomy plus MCB is feasible and effective in preventing 2nd LR with an OS rate at least equivalent to those achieved with salvage mastectomy.

Adjuvant treatment of premenopausal women with endocrine-responsive early breast cancer: design of the TEXT and SOFT trials

OBJECTIVES In 2003 the International Breast Cancer Study Group (IBCSG) initiated the TEXT and SOFT randomized phase III trials to answer two questions concerning adjuvant treatment for premenopausal women with endocrine-responsive early breast cancer: 1-What is the role of aromatase inhibitors (AI) for women treated with ovarian function suppression (OFS)? 2-What is the role of OFS for women who remain premenopausal and are treated with tamoxifen? METHODS TEXT randomized patients to receive exemestane or tamoxifen with OFS. SOFT randomized patients to receive exemestane with OFS, tamoxifen with OFS, or tamoxifen alone. Treatment was for 5 years from randomization. RESULTS TEXT and SOFT successfully met their enrollment goals in 2011. The 5738 enrolled women had lower-risk disease and lower observed disease-free survival (DFS) event rates than anticipated. Consequently, 7 and 13 additional years of follow-up for TEXT and SOFT, respectively, were required to reach the targeted DFS events (median follow-up about 10.5 and 15 years). To provide timely answers, protocol amendments in 2011 specified analyses based on chronological time and median follow-up. To assess the AI question, exemestane + OFS versus tamoxifen + OFS, a combined analysis of TEXT and SOFT became the primary analysis (n = 4717). The OFS question became the primary analysis from SOFT, assessing the unique comparison of tamoxifen + OFS versus tamoxifen alone (n = 2045). The first reports are anticipated in mid- and late-2014. CONCLUSIONS We present the original designs of TEXT and SOFT and adaptations to ensure timely answers to two questions concerning optimal adjuvant endocrine treatment for premenopausal women with endocrine-responsive breast cancer. Trial Registration TEXT: Clinicaltrials.govNCT00066703 SOFT: Clinicaltrials.govNCT00066690.

Adjuvant pegylated liposomal doxorubicin for older women with endocrine nonresponsive breast cancer who are NOT suitable for a "standard chemotherapy regimen": the CASA randomized trial

There is no optimal treatment for breast cancers lacking estrogen (ER) and progesterone (PgR) receptors in elderly women with co-morbidities that prevent use of "standard chemotherapy regimens" such as AC or CMF. The CASA trial studied pegylated liposomal doxorubicin (PLD) and low dose, metronomic cyclophosphamide + methotrexate (CM) for older (>65), vulnerable women with operable, ER and PgR-negative breast cancer. After two years the trial closed early, due to slow and inadequate accrual, with 77 patients (38:PLD, 36:CM, 3:nil). Sixty-eight percent completed PLD; 83% completed CM (both 16 weeks). Patients on PLD reported worse quality of life, cognitive and physical functioning than non-PLD regimens (primarily CM). At a median follow-up of 42 months, 81% of randomized patients remained free of any breast cancer recurrence. Based on our limited experience, PLD and CM may be reasonable options for further study for elderly vulnerable patients with endocrine nonresponsive breast cancer.

Exemestane Versus Anastrozole in Postmenopausal Women With Early Breast Cancer: NCIC CTG MA.27—A Randomized Controlled Phase III Trial

PURPOSE In patients with hormone-dependent postmenopausal breast cancer, standard adjuvant therapy involves 5 years of the nonsteroidal aromatase inhibitors anastrozole and letrozole. The steroidal inhibitor exemestane is partially non-cross-resistant with nonsteroidal aromatase inhibitors and is a mild androgen and could prove superior to anastrozole regarding efficacy and toxicity, specifically with less bone loss. PATIENTS AND METHODS We designed an open-label, randomized, phase III trial of 5 years of exemestane versus anastrozole with a two-sided test of superiority to detect a 2.4% improvement with exemestane in 5-year event-free survival (EFS). Secondary objectives included assessment of overall survival, distant disease-free survival, incidence of contralateral new primary breast cancer, and safety. RESULTS In the study, 7,576 women (median age, 64.1 years) were enrolled. At median follow-up of 4.1 years, 4-year EFS was 91% for exemestane and 91.2% for anastrozole (stratified hazard ratio, 1.02; 95% CI, 0.87 to 1.18; P = .85). Overall, distant disease-free survival and disease-specific survival were also similar. In all, 31.6% of patients discontinued treatment as a result of adverse effects, concomitant disease, or study refusal. Osteoporosis/osteopenia, hypertriglyceridemia, vaginal bleeding, and hypercholesterolemia were less frequent on exemestane, whereas mild liver function abnormalities and rare episodes of atrial fibrillation were less frequent on anastrozole. Vasomotor and musculoskeletal symptoms were similar between arms. CONCLUSION This first comparison of steroidal and nonsteroidal classes of aromatase inhibitors showed neither to be superior in terms of breast cancer outcomes as 5-year initial adjuvant therapy for postmenopausal breast cancer by two-way test. Less toxicity on bone is compatible with one hypothesis behind MA.27 but requires confirmation. Exemestane should be considered another option as up-front adjuvant therapy for postmenopausal hormone receptor-positive breast cancer.