Susan Minton

Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer

Background:Histone deacetylases (HDACs) are crucial components of the oestrogen receptor (ER) transcriptional complex. Preclinically, HDAC inhibitors can reverse tamoxifen/aromatase inhibitor resistance in hormone receptor-positive breast cancer. This concept was examined in a phase II combination trial with correlative end points.Methods:Patients with ER-positive metastatic breast cancer progressing on endocrine therapy were treated with 400 mg of vorinostat daily for 3 of 4 weeks and 20 mg tamoxifen daily, continuously. Histone acetylation and HDAC2 expression in peripheral blood mononuclear cells were also evaluated.Results:In all, 43 patients (median age 56 years (31–71)) were treated, 25 (58%) received prior adjuvant tamoxifen, 29 (67%) failed one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by Response Evaluation Criteria in Solid Tumours criteria was 19% and the clinical benefit rate (response or stable disease >24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1–12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response.Conclusion:The combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. Correlative studies suggest that HDAC2 expression is a predictive marker and histone hyperacetylation is a useful pharmacodynamic marker for the efficacy of this combination.

Activation of stat3 in primary tumors from high-risk breast cancer patients is associated with elevated levels of activated SRC and survivin expression.

Purpose: Constitutive activation of signal transducer and activator of transcription 3 (Stat3) protein has been observed in a wide variety of tumors, including breast cancer, and contributes to oncogenesis at least in part by prevention of apoptosis. In a study of 45 patients with high-risk breast cancer enrolled in a phase II neoadjuvant chemotherapy trial with docetaxel and doxorubicin, we evaluated the levels of Stat3 activation and potentially associated molecular biomarkers in invasive breast carcinoma compared with matched nonneoplastic tissues. Experimental Design: Using immunohistochemistry and image analysis, we quantified the levels of phospho-Stat3 (pY-Stat3), phospho-Src (pY-Src), epidermal growth factor receptor, HER2/neu, Ki-67, estrogen receptor, Bcl-2, Bcl-xL, Survivin, and apoptosis in formalin-fixed, paraffin-embedded sections from invasive carcinomas and their paired nonneoplastic parenchyma. The levels of molecular biomarkers in nonneoplastic and tumor tissues were analyzed as continuous variables for statistically significant correlations. Results: Levels of activated pY-Stat3 and pY-Src measured by immunohistochemistry were significantly higher in invasive carcinoma than in nonneoplastic tissue (P < 0.001). In tumors, elevated levels of pY-Stat3 correlated with those of pY-Src and Survivin. Levels of pY-Stat3 were higher in partial pathologic responders than in complete pathologic responders. In partial pathologic responders, pY-Stat3 levels correlated with Survivin expression. Conclusions: Our findings suggest important roles for elevated activities of Stat3 and Src, as well as Survivin expression, in malignant progression of breast cancer. Furthermore, elevated Stat3 activity correlates inversely with complete pathologic response. These findings suggest that specific Stat3 or Src inhibitors could offer clinical benefits to patients with breast cancer.

Phase I Trial of Histone Deacetylase Inhibition by Valproic Acid Followed by the Topoisomerase II Inhibitor Epirubicin in Advanced Solid Tumors: A Clinical and Translational Study

PURPOSE To determine the safety, toxicity, and maximum-tolerated dose of a sequence-specific combination of the histone deacetylase inhibitor (HDACi), valproic acid (VPA), and epirubicin in solid tumor malignancies and to define the clinical feasibility of VPA as an HDACi. PATIENTS AND METHODS Patients were treated with increasing doses of VPA (days 1 through 3) followed by epirubicin (day 3) in 3-week cycles. The study evaluated pharmacokinetic and pharmacodynamic end points, toxicities, and tumor response. RESULTS Forty-eight patients were enrolled, and 44 received at least one cycle of therapy. Patients (median age, 54 years; range, 39 to 78 years) received the following doses of VPA: 15, 30, 45, 60, 75, 90, 100, 120, 140, and 160 mg/kg/d. Dose-limiting toxicities were somnolence (n = 1), confusion (n = 3), and febrile neutropenia (n = 1). No exacerbation of epirubicin-related toxicities was observed. Partial responses were seen across different tumor types in nine patients (22%), and stable disease/minor responses were seen in 16 patients (39%), despite a median number of three prior regimens (range, zero to 10 prior regimens). Patients received a median number of four treatment cycles (range, one to 10 cycles), and treatment was stopped after reaching maximal epirubicin doses rather than progression in 13 (32%) of 41 patients patients. Total and free VPA plasma concentrations increased linearly with dose and correlated with histone acetylation in peripheral-blood mononuclear cells. CONCLUSION The maximum-tolerated dose and recommended phase II dose was VPA 140 mg/kg/d for 48 hours followed by epirubicin 100 mg/m2. Sustained plasma concentrations of VPA exceeding those required for in vitro synergy were achieved with acceptable toxicity. Noteworthy antitumor activity was observed in heavily pretreated patients and historically anthracycline-resistant tumors.

Randomized Trial Using Gonadotropin-Releasing Hormone Agonist Triptorelin for the Preservation of Ovarian Function During (Neo)Adjuvant Chemotherapy for Breast Cancer

PURPOSE Chemotherapy-induced amenorrhea is a serious concern for women undergoing cancer therapy. This prospective randomized trial evaluated the use of gonadotropin-releasing hormone (GnRH) analog triptorelin to preserve ovarian function in women treated with chemotherapy for early-stage breast cancer. PATIENTS AND METHODS Premenopausal women age 44 years or younger were randomly assigned to receive either triptorelin or no triptorelin during (neo)adjuvant chemotherapy and were further stratified by age (< 35, 35 to 39, > 39 years), estrogen receptor status, and chemotherapy regimen. Objectives included the resumption of menses and serial monitoring of follicle-stimulating hormone (FSH) and inhibin A and B levels. RESULTS Targeted for 124 patients with a planned 5-year follow-up, the trial was stopped for futility after 49 patients were enrolled (median age, 39 years; range, 21 to 43 years); 47 patients were treated according to assigned groups with four cycles of adriamycin plus cyclophosphamide alone or followed by four cycles of paclitaxel or six cycles of fluorouracil, epirubicin, and cyclophosphamide. Menstruation resumed in 19 (90%) of 21 patients in the control group and in 23 (88%) of 26 in the triptorelin group (P= .36). Menses returned after a median of 5.8 months (range, 1 to 19 months) after completion of chemotherapy in the triptorelin versus 5.0 months (range, 0 to 28 months) in the control arm (P= .58). Two patients (age 26 and 35 years at random assignment) in the control group had spontaneous pregnancies with term deliveries. FSH and inhibin B levels correlated with menstrual status. CONCLUSION When stratified for age, estrogen receptor status, and treatment regimen, amenorrhea rates on triptorelin were comparable to those seen in the control group.

Chemotherapy-induced amenorrhea and fertility in women undergoing adjuvant treatment for breast cancer.

BACKGROUND The majority of women diagnosed with early-stage breast cancer have an excellent long-term prognosis, but many will undergo temporary or permanent chemotherapy-induced amenorrhea. METHODS While breast cancer is more common in older women, about 1 in 200 women under the age of 40 is at risk to develop breast cancer. Many of these women benefit from chemotherapy but are afraid to risk the opportunity to bear children. The authors review the current studies on the impact of adjuvant chemotherapy on amenorrhea and fertility in women with breast cancer. RESULTS The likelihood of amenorrhea is based on the specific adjuvant chemotherapy regimen administered and the age of the patient. Future childbirth is a viable option for women treated for breast cancer at an early stage. While the use of tamoxifen as a hormonal therapy in premenopausal breast cancer is now the standard of care, no conclusive data confirm the benefit of GnRH agonists in adjuvant therapy after treatment with chemotherapy followed by tamoxifen. CONCLUSIONS As more women over the age of 35 consider pregnancy, fertility issues are becoming important areas of investigation for the adjuvant treatment of breast cancer. Whether chemotherapy-induced amenorrhea has a prognostic effect remains unclear, and further studies are warranted.

Phase I trial of vorinostat and doxorubicin in solid tumours: histone deacetylase 2 expression as a predictive marker

Background:Histone deacetylase inhibitors (HDACi) can sensitise cancer cells to topoisomerase inhibitors by increasing their access and binding to DNA.Methods:This phase I trial was designed to determine the toxicity profile, tolerability, and recommended phase II dose of escalating doses of the HDACi vorinostat, with weekly doxorubicin.Results:In total, 32 patients were treated; vorinostat was dosed at 400, 600, 800, or 1000 mg day−1 on days 1–3, followed by doxorubicin (20 mg m−2) on day 3 for 3 of 4 weeks. Maximal tolerated dose was determined to be 800 mg day−1 of vorinostat. Dose-limiting toxicities were grade 3 nausea/vomiting (two out of six) and fatigue (one out of six) at 1000 mg day−1. Non-dose-limiting grade 3/4 toxicities included haematological toxicity and venous thromboembolism. Antitumor activity in 24 evaluable patients included two partial responses (breast and prostate cancer). Two patients with melanoma had stable disease for ⩾8 months. Histone hyperacetylation changes in peripheral blood mononuclear and tumour cells were comparable. Histone hyperacetylation seemed to correlate with pre-treatment HDAC2 expression.Conclusion:These findings suggest that vorinostat can be combined with weekly doxorubicin in this schedule at a dose of 800 mg day−1. The HDAC2 expression may be a marker predictive of HDAC inhibition. Antitumor activity of this regimen in breast cancer, prostate cancer, and melanoma seems interesting.

A pilot trial of paroxetine for the treatment of hot flashes and associated symptoms in women with breast cancer.

Abrupt onset of hot flashes poses a significant problem for women treated with chemotherapy for breast cancer. Alternatives to hormone replacement, such as the use of the selective serotonin re-uptake inhibitor (SSRI) paroxetine hydrochloride, are being explored as therapies for hot flashes in this patient population. The present study investigated the efficacy of paroxetine for the treatment of hot flashes and associated symptoms in women with breast cancer. This study included 13 patients who were seen in the Psychosocial Clinic at Moffitt Cancer Center. They were referred by their medical oncologist after reporting complaints of significant difficulty with hot flashes. Baseline questionnaires were completed and a structured diagnostic interview for clinical depression was conducted, all of which were repeated 5 weeks after the paroxetine 20 mg daily was started. Significant improvements were seen in the ratings of hot flash severity (P = 0.002). In addition, significant improvements were observed in general, emotional, and mental fatigue. Rates of clinically significant depressive symptomatology also decreased and sleep quality improved significantly as well. Finally, the incidence of clinical depression improved from 39% at baseline to 8% after treatment. These preliminary data suggest that the antidepressant paroxetine can be helpful in the treatment of hot flashes and associated fatigue, sleep disturbance, and depression in women with breast cancer treated with chemotherapy. Further controlled studies are needed to more fully evaluate the efficacy of the SSRIs for hot flashes in women with breast cancer.

Clinical and Biological Effects of Valproic Acid as a Histone Deacetylase Inhibitor on Tumor and Surrogate Tissues: Phase I/II Trial of Valproic acid and Epirubicin/FEC

Purpose: The aim was to study the biological and molecular effects of the histone deacetylase (HDAC) inhibitor, valproic acid, in patients with solid tumor malignancies. Experimental Design: A phase I dose escalation of valproic acid given on days 1 to 3 followed by epirubicin (day 3) was followed by a dose expansion of valproic acid combined with 5-fluorouracil, epirubicin, and cyclophosphamide (FEC100). Pharmacodynamic and pharmacokinetic studies entailed valproic acid and epirubicin plasma levels and their interaction, the effects of valproic acid on histone acetylation in peripheral blood mononuclear cells (PBMC) and tumor cells at baseline and day 3, and baseline expression of HDAC2 and HDAC6 as therapeutic targets. Results: Forty-four patients were enrolled in the phase I part, with a disease-specific cohort expansion of 15 breast cancer patients (median age, 55 years; range, 28-66 years) receiving 120 mg/kg/day valproic acid followed by FEC100. Partial responses were seen in 9 of 41 (22%) patients during the phase I part. Objective responses were seen in 9 of 14 (64%) evaluable patients at the dose expansion with a median number of 6 administered cycles. Predominant toxicities were valproic acid–associated somnolence and epirubicin-induced myelosuppression. Valproic acid plasma levels were associated with short-term, reversible depletion of WBC and neutrophils within 48 hours. Histone acetylation in tumor samples and in PBMCs correlated with valproic acid levels and was further linked to baseline HDAC2 but not to HDAC6 expression. Conclusion: Valproic acid is a clinically relevant HDAC inhibitor, and PBMCs may serve as a surrogate for tumor histone acetylation in solid tumor malignancies. HDAC2 should be further considered as a relevant therapeutic target.

Sentinel node biopsy before neoadjuvant chemotherapy for determining axillary status and treatment prognosis in locally advanced breast cancer

BackgroundTreatment of locally advanced breast cancer with neoadjuvant chemotherapy assesses an in vivo tumor response while increasing breast conservation. Axillary clearance of nodal disease after treatment defines prognostic stratification. Our study objective was to show that sentinel node staging before treatment can optimize posttreatment prognostic stratification in clinically N0 patients.MethodsEighty-nine patients with locally advanced breast cancer were treated with neoadjuvant chemotherapy. Of these, 42 (47%) clinically palpable or image-detected nodes (cN+) were histologically confirmed before treatment (group 1), and 47 (53%) patients without palpable lymph nodes (cN0) had a sentinel lymph node (SLN) biopsy before treatment (group 2). Survival analysis was conducted with the Kaplan-Meier method.ResultsIn groups 1 and 2, 82 (92%) of 89 patients had node-positive disease before treatment. Seven (8%) of 89 had negative SLNs and no completion axillary lymph node dissection, 24 (27%) patients had a complete pathologic axillary response (pCRAX; 11 [26%] of 42 in group 1 and 13 [33%] of 40 in group 2), and 58 (65%) of 89 had residual disease in the axilla. Breast-conserving therapy was applied to 27 (30%) of 89 patients. The seven SLN-negative patients had no axillary recurrence at 25 months, and pCRAX patients had a significantly higher overall survival than patients with residual disease.ConclusionsThis study validates the prognostic stratification of patients with a complete pathologic axillary response to neoadjuvant chemotherapy. The addition of SLN biopsy to cN0 patients before treatment increased accurate nodal staging by 53%, eliminated completion axillary lymph node dissection in 15%, and demonstrated an improved prognosis in 28% of pCRAX patients. SLN biopsy before treatment provides accurate staging of cN0 patients; allows acquisition of standard treatment markers, prognostic biomarkers, and microarray analysis; and affords prognostic stratification after treatment.