Ritonavir inhibits intratumoral docetaxel metabolism and enhances docetaxel antitumor activity in an immunocompetent mouse breast cancer model

Docetaxel (Taxotere(®) ) is currently used intravenously as an anticancer agent and is primarily metabolized by Cytochrome P450 3A (CYP3A). The HIV protease inhibitor ritonavir, a strong CYP3A4 inhibitor, decreased first-pass metabolism of orally administered docetaxel. Anticancer effects of ritonavir itself have also been described. We here aimed to test whether ritonavir co-administration could decrease intratumoral metabolism of intravenously administered docetaxel and thus increase the antitumor activity of docetaxel in an orthotopic, immunocompetent mouse model for breast cancer. Spontaneously arising K14cre;Brca1(F/F) ;p53(F/F) mouse mammary tumors were orthotopically implanted in syngeneic mice lacking Cyp3a (Cyp3a(-/-) ) to limit ritonavir effects on systemic docetaxel clearance. Over 3 weeks, docetaxel (20 mg/kg) was administered intravenously once weekly, with or without ritonavir (12.5 mg/kg) administered orally for 5 days per week. Untreated mice were used as control for tumor growth. Ritonavir treatment alone did not significantly affect the median time of survival (14 vs. 10 days). Median time of survival in docetaxel-treated mice was 54 days. Ritonavir co-treatment significantly increased this to 66 days, and substantially reduced relative average tumor size, without altering tumor histology. Concentrations of the major docetaxel metabolite M2 in tumor tissue were reduced by ritonavir co-administration, whereas tumor RNA expression of Cyp3a was unaltered. In this breast cancer model, we observed no direct antitumor effect of ritonavir alone, but we found enhanced efficacy of docetaxel treatment when combined with ritonavir. Our data, therefore, suggest that decreased docetaxel metabolism inside the tumor as a result of Cyp3a inhibition contributes to increased antitumor activity.

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.

Positron emission mammography in the diagnosis of breast cancer. Is maximum PEM uptake value a valuable threshold for malignant breast cancer detection?

AIM To evaluate the diagnostic value (sensitivity, specificity) of positron emission mammography (PEM) in a single site non-interventional study using the maximum PEM uptake value (PUVmax). PATIENTS, METHODS In a singlesite, non-interventional study, 108 patients (107 women, 1 man) with a total of 151 suspected lesions were scanned with a PEM Flex Solo II (Naviscan) at 90 min p.i. with 3.5 MBq 18F-FDG per kg of body weight. In this ROI(region of interest)-based analysis, maximum PEM uptake value (PUV) was determined in lesions, tumours (PUVmaxtumour), benign lesions (PUVmaxnormal breast) and also in healthy tissues on the contralateral side (PUVmaxcontralateral breast). These values were compared and contrasted. In addition, the ratios of PUVmaxtumour / PUVmaxcontralateral breast and PUVmaxnormal breast / PUVmaxcontralateral breast were compared. The image data were interpreted independently by two experienced nuclear medicine physicians and compared with histology in cases of suspected carcinoma. RESULTS Based on a criteria of PUV>1.9, 31 out of 151 lesions in the patient cohort were found to be malignant (21%). A mean PUVmaxtumour of 3.78 ± 2.47 was identified in malignant tumours, while a mean PUVmaxnormal breast of 1.17 ± 0.37 was reported in the glandular tissue of the healthy breast, with the difference being statistically significant (p < 0.001). Similarly, the mean ratio between tumour and healthy glandular tissue in breast cancer patients (3.15 ± 1.58) was found to be significantly higher than the ratio for benign lesions (1.17 ± 0.41, p < 0.001). CONCLUSION PEM is capable of differentiating breast tumours from benign lesions with 100% sensitivity along with a high specificity of 96%, when a threshold of PUVmax >1.9 is applied.

Predicting 5- and 10-year survival in older women with early-stage breast cancer: self-rated health and walking ability.

OBJECTIVES To determine life expectancy for older women with breast cancer. DESIGN Prospective longitudinal study with 10 years of follow-up data. SETTING Hospitals or collaborating tumor registries in four geographic regions (Los Angeles, California; Minnesota; North Carolina; Rhode Island). PARTICIPANTS Women aged 65 and older at time of breast cancer diagnosis with Stage I to IIIA disease with measures of self-rated health (SRH) and walking ability at baseline (N = 615; 17% aged ≥80, 52% Stage I, 58% with ≥2 comorbidities). MEASUREMENTS Baseline SRH, baseline self-reported walking ability, all-cause and breast cancer-specific estimated probability of 5- and 10-year survival. RESULTS At the time of breast cancer diagnosis, 39% of women reported poor SRH, and 28% reported limited ability to walk several blocks. The all-cause survival curves appear to separate after approximately 3 years, and the difference in survival probability between those with low SRH and limited walking ability and those with high SRH and no walking ability limitation was significant (0.708 vs 0.855 at 5 years, P ≤ .001; 0.300 vs 0.648 at 10 years, P < .001). There were no differences between the groups in breast cancer-specific survival at 5 and 10 years (P = .66 at 5 years, P = .16 at 10 years). CONCLUSION The combination of low SRH and limited ability to walk several blocks at diagnosis is an important predictor of worse all-cause survival at 5 and 10 years. These self-report measures easily assessed in clinical practice may be an effective strategy to improve treatment decision-making in older adults with cancer.

Fertility preservation options in breast cancer patients.

The purpose of this review is to analyse current options for fertility preservation in young women with breast cancer (BC). Considering an increasing number of BC survivors, owing to improvements in cancer treatment and delaying of childbearing, fertility preservation appears to be an important issue. Current fertility preservation options in BC survivors range from well-established standard techniques to experimental or investigational interventions. Among the standard options, random-start ovarian stimulation protocol represents a new technique, which significantly decreases the total time of the in vitro fertilisation cycle. However, in patients with oestrogen-sensitive tumours, stimulation protocols using aromatase inhibitors are currently preferred over tamoxifen regimens. Cryopreservation of embryos and oocytes are nowadays deemed the most successful techniques for fertility preservation in BC patients. GnRH agonists during chemotherapy represent an experimental method for fertility preservation due to conflicting long-term outcome results regarding its safety and efficacy. Cryopreservation of ovarian tissue, in vitro maturation of immature oocytes and other strategies are considered experimental and should only be offered within the context of a clinical trial. An early pretreatment referral to reproductive endocrinologists and oncologists should be suggested to young BC women at risk of infertility, concerning the risks and benefits of fertility preservation options.

Doxorubicin Affects Expression of Proteins of Neuronal Pathways in MCF-7 Breast Cancer Cells.

In the present article, we report on the semi-quantitative proteome analysis and related changes in protein expression of the MCF-7 breast cancer cell line following treatment with doxorubicin, using the precursor acquisition independent from ion count (PAcIFIC) mass spectrometry method. PAcIFIC represents a cost-effective and easy-to-use proteomics approach, enabling for deep proteome sequencing with minimal sample handling. The acquired proteomic data sets were searched for regulated Reactome pathways and Gene Ontology annotation terms using a new algorithm (SetRank). Using this approach, we identified pathways with significant changes (≤0.05), such as chromatin organization, DNA binding, embryo development, condensed chromosome, sequence-specific DNA binding, response to oxidative stress and response to toxin, as well as others. These sets of pathways are already well-described as being susceptible to chemotherapeutic drugs. Additionally, we found pathways related to neuron development, such as central nervous system neuron differentiation, neuron projection membrane and SNAP receptor activity. These later pathways might indicate biological mechanisms on the molecular level causing the known side-effect of doxorubicin chemotherapy, characterized as cognitive impairment, also called 'chemo brain'. Mass spectrometry data are available via ProteomeXchange with identifier PXD002998.

TUMOR PROGRESSION: ANALYSIS OF THE INSTABILITY OF THE METASTATIC PHENOTYPE, SENSITIVITY TO RADIATION AND CHEMOTHERAPY (PHENOTYPIC DRIFT, BREAST CANCER)

The major complications for tumor therapy are (i) tumor spread (metastasis); (ii) the mixed nature of tumors (heterogeneity); and (iii) the capacity of tumors to evolve (progress). To study these tumor characteristics, the rat 13762NF mammary adenocarcinoma was cloned and studied for metastatic properties and sensitivities to therapy (chemotherapy, radiation and hyperthermia). The cell clones were heterogeneous and no correlation between metastatic potential and therapeutic sensitivities was observed. Further, these phenotypes were unstable during passage in vitro; yet, the changes were clone dependent and reproducible using different cryoprotected cell stocks. To understand the phenotypic instability, subclones were isolated from low and high passage cell clones. Each subclone possessed a unique composite phenotype. Again, no apparent correlation was seen between metastatic potential and sensitivity to therapy. The results demonstrated that (1) tumor cells are heterogeneous for multiple phenotypes; (2) tumor cells are unstable for multiple phenotypes; (3) the magnitude, direction and time of occurrence of phenotypic drift is clone dependent; (4) the sensitivity of cell clones to ionizing radiation (gamma or heat) and chemotherapy agents is independent of their metastatic potential; (5) shifts in metastatic potential and sensitivity to therapy may occur simultaneously but are not linked; and (6) tumor cells independently diverge to form several subpopulations with unique phenotypic profiles. ^

Two prognostic factors in breast cancer: Novel functions of EGFR and beta-catenin

In this study, we demonstrated the novel functions of two important prognostic markers in breast cancer, EGFR and b -catenin in proliferation and/or other transformation phenotype. ^ First we demonstrated that EGFR could be detected in the nucleus in highly proliferating tissues, including primary breast cancer samples and a breast cancer cell line. We found that EGFR contained a strong transactivation domain, complexed with an AT-rich consensus DNA sequence and activated promoters containing this sequence, including cyclin D1 promoter. Therefore, EGFR may function as a transcription factor to activate genes required for highly proliferating activity such as cyclin D1 in breast cancer. ^ In the second part of this study, we identified b -catenin as an important prognostic factor in breast cancer. We found that cyclin D1 was one of the genes regulated by b -catenin in breast cancer cells. The transactivation activity of b -catenin correlated significantly with cyclin D1 expression in both breast cancer cell lines and in breast cancer patient samples, in which high b -catenin activity correlated with poor prognosis of the patients. Moreover, blockage of b -catenin activity significantly inhibited transformation phenotypes in breast cancer cells. Therefore, our results indicate that b -catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy. ^

Primary lung cancer after breast cancer: The role of radiation therapy and cigarette smoking

The magnitude of the interaction between cigarette smoking, radiation therapy, and primary lung cancer after breast cancer remains unresolved. This case control study further examines the main and joint effects of cigarette smoking and radiation therapy (XRT) among breast cancer patients who subsequently developed primary lung cancer, at The University of Texas M. D. Anderson Cancer Center (MDACC) in Houston, Texas. Cases (n = 280) were women diagnosed with primary lung cancer between 1955 and 1970, between 30–89 years of age, who had a prior history of breast cancer, and were U.S. residents. Controls (n = 300) were randomly selected from 37,000 breast cancer patients at MDACC and frequency matched to cases on age at diagnosis (in 5-year strata), ethnicity, year of breast cancer diagnosis (in 5-year strata), and had survived at least as long as the time interval for lung cancer diagnosis in the cases. Stratified analysis and unconditional logistic regression modeling were used to calculate the main and joint effects of cigarette smoking and radiation treatment on lung cancer risk. Medical record review yielded smoking information on 93% of cases and 84% of controls, and among cases 45% received XRT versus 44% of controls. Smoking increased the odds of lung cancer in women who did not receive XRT (OR = 6.0, 95%CI, 3.5–10.1) whereas XRT was not associated with increased odds (OR = 0.5, 95%CI, 0.2–1.1) in women who did not smoke. Overall the odds ratio for both XRT and smoking together compared with neither exposure was 9.00 (9 5% CI, 5.1–15.9). Similarly, when stratifying on laterality of the lung cancer in relation to the breast cancer, and when the time interval between breast and lung cancers was >10 years, there was an increased odds for both smoking and XRT together for lung cancers on the same side as the breast cancer (ipsilateral) (OR = 11.5, 95% CI, 4.9–27.8) and lung cancers on the opposite side of the breast cancer (contralateral) (OR= 9.6, 95% CI, 2.9–0.9). After 20 years the odds for the ipsilateral lung were even more pronounced (OR = 19.2, 95% CI, 4.2–88.4) compared to the contralateral lung (OR = 2.6, 95% CI, 0.2–2.1). In conclusion, smoking was a significant independent risk factor for lung cancer after breast cancer. Moreover, a greater than multiplicative effect was observed with smoking and XRT combined being especially evident after 10 years for both the ipsilateral and contralateral lung and after 20 years for the ipsilateral lung. ^

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

Prime and boost vaccination against HER2/neu in breast cancer

Breast cancer is the most common cancer among women with approximately 180,000 new cases being diagnosed yearly in the United States (1). HER2/neu gene amplification and subsequent protein overexpression is found in 20–30% of breast cancer patients and can lead to the promotion of various metastasis-related properties (2–4) and/or resistance to cancer therapies such as chemotherapy and radiation (5). ^ The protein product of the HER2/neu gene, p185, is a proven target for immunological therapy. Recently, passive immunotherapy with the monoclonal antibody Trastuzumab® has validated an immunological approach to HER2/neu+ breast cancer. Immunity to HER2/ neu, when found in breast cancer patients, is of low magnitude. Vaccination-induced HER2/neu-specific antibodies and HER2/neu-specific cytotoxic T cells could result in long-lived immunity with therapeutic benefit. Many features of DNA vaccines and attenuated viral vectors may contribute to the efficacy of prime-boost vaccination. In particular, vaccines capable of eliciting strong cell-mediated immunity are thought to hold the greatest promise for control of cancer (6–9). ^ To optimize cellular immunization to HER2/neu in my study, the HER2/neu gene was presented to the immune system using a priming vector followed by a second vector used as the boost. In both animals and humans, priming with DNA and boosting with a poxviruses, vaccinia or canarypox appears to be particularly promising for induction of a broad immune responses (10). ^ I tested three gene vaccines encoding the HER2/neu gene: (1) a plasmid, SINCP, that contains part of the genome of Sindbis virus; (2) Viral Replicon Particles (VRP) of Venezuela Equine Encephalitis virus (VEE) and (3) E1/E2a-deleted human Type 5 Adenovirus. In SINCP and the VRP, the caspid and envelope genes of the virus were deleted and replaced with the gene for HER2/neu. SINCP-neu, VRP- neu and Adeno-neu when used alone were effective vaccines protecting healthy mice from challenge with a breast cancer cell line injected in the mammary fat pad or injected i.v. to induce experimental lung metastasis. However, SINCP-neu, VRP-neu or Adeno-neu when used alone were not able to prolong survival of mice in therapeutic models in which vaccination occurred after injection of a breast cancer cell line. ^ When the vaccines were combined in a mixed regimen of a SINCP- neu prime VRP-neu or Adeno-neu boost, there was a significant difference in tumor growth and survival in the therapeutic vaccine models. In vitro assays demonstrated that vaccination with each of the three vaccines induced IgG specific for p185, the gene product of HER2/neu, induced p185-specific T lymphocytes, as measured by tetramer analysis. Vaccination also induced intracellular INF-γ and a positive ELISPOT assay. These findings indicate that SINCP-neu, VRP-neu and Adeno-neu, used alone or in combination, may have clinical potential as adjuvant immunotherapy for the treatment of HER2/neu-expressing tumors. ^

Interrelationships between the structural and functional aspects of social relationships, comorbidity and health related quality of life in breast cancer survivors

It is widely acknowledged in theoretical and empirical literature that social relationships, comprising of structural measures (social networks) and functional measures (perceived social support) have an undeniable effect on health outcomes. However, the actual mechanism of this effect has yet to be clearly understood or explicated. In addition, comorbidity is found to adversely affect social relationships and health related quality of life (a valued outcome measure in cancer patients and survivors). ^ This cross sectional study uses selected baseline data (N=3088) from the Women's Healthy Eating and Living (WHEL) study. Lisrel 8.72 was used for the latent variable structural equation modeling. Due to the ordinal nature of the data, Weighted Least Squares (WLS) method of estimation using Asymptotic Distribution Free covariance matrices was chosen for this analysis. The primary exogenous predictor variables are Social Networks and Comorbidity; Perceived Social Support is the endogenous predictor variable. Three dimensions of HRQoL, physical, mental and satisfaction with current quality of life were the outcome variables. ^ This study hypothesizes and tests the mechanism and pathways between comorbidity, social relationships and HRQoL using latent variable structural equation modeling. After testing the measurement models of social networks and perceived social support, a structural model hypothesizing associations between the latent exogenous and endogenous variables was tested. The results of the study after listwise deletion (N=2131) mostly confirmed the hypothesized relationships (TLI, CFI >0.95, RMSEA = 0.05, p=0.15). Comorbidity was adversely associated with all three HRQoL outcomes. Strong ties were negatively associated with perceived social support; social network had a strong positive association with perceived social support, which served as a mediator between social networks and HRQoL. Mental health quality of life was the most adversely affected by the predictor variables. ^ This study is a preliminary look at the integration of structural and functional measures of social relationships, comorbidity and three HRQoL indicators using LVSEM. Developing stronger social networks and forming supportive relationships is beneficial for health outcomes such as HRQoL of cancer survivors. Thus, the medical community treating cancer survivors as well as the survivor's social networks need to be informed and cognizant of these possible relationships. ^