Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

BACKGROUND The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.

Development of a Tumour Growth Inhibition Model to Elucidate the Effects of Ritonavir on Intratumoural Metabolism and Anti-tumour Effect of Docetaxel in a Mouse Model for Hereditary Breast Cancer

In a mouse tumour model for hereditary breast cancer, we previously explored the anti-cancer effects of docetaxel, ritonavir and the combination of both and studied the effect of ritonavir on the intratumoural concentration of docetaxel. The objective of the current study was to apply pharmacokinetic (PK)-pharmacodynamic (PD) modelling on this previous study to further elucidate and quantify the effects of docetaxel when co-administered with ritonavir. PK models of docetaxel and ritonavir in plasma and in tumour were developed. The effect of ritonavir on docetaxel concentration in the systemic circulation of Cyp3a knock-out mice and in the implanted tumour (with inherent Cyp3a expression) was studied, respectively. Subsequently, we designed a tumour growth inhibition model that included the inhibitory effects of both docetaxel and ritonavir. Ritonavir decreased docetaxel systemic clearance with 8% (relative standard error 0.4%) in the co-treated group compared to that in the docetaxel only-treated group. The docetaxel concentration in tumour tissues was significantly increased by ritonavir with mean area under the concentration-time curve 2.5-fold higher when combined with ritonavir. Observed tumour volume profiles in mice could be properly described by the PK/PD model. In the co-treated group, the enhanced anti-tumour effect was mainly due to increased docetaxel tumour concentration; however, we demonstrated a small but significant anti-tumour effect of ritonavir addition (p value <0.001). In conclusion, we showed that the increased anti-tumour effect observed when docetaxel is combined with ritonavir is mainly caused by enhanced docetaxel tumour concentration and to a minor extent by a direct anti-tumour effect of ritonavir.

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.

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. ^

The effect of African Americans' ethnographic paradigms and cultural explanatory model of breast cancer in framing the male partner's attitudes and behaviors about the woman's breast cancer diagnosis

The disparate burden of breast cancer-related morbidity and mortality experienced by African American women compared with women of other races is a topic of intense debate in the medical and public health arenas. The anomaly is consistently attributed to the fact that at diagnosis, a large proportion of African American women have advanced-stage disease. Extensive research has documented the impacts of cultural factors and of socioeconomic factors in shaping African American women's breast-health practices; however, there is another factor of a more subtle influence that might have some role in establishing these women's vulnerability to this disease: the lack of or perceived lack of partner support. Themes expressed in the research literature reflect that many African American breast cancer patients and survivors consider their male partners as being apathetic and nonsupportive. ^ The purpose of this study was to learn how African American couples' ethnographic paradigms and cultural explanatory model of breast cancer frame the male partners' responses to the women's diagnosis and to assess his ability to cope and willingness to adapt to the subsequent challenges. The goal of the study was to determine whether these men's coping and adaptation skills positively or negatively affect the women's self-care attitudes and behaviors. ^ This study involved 4 African American couples in which the woman was a breast cancer survivor. Participants were recruited through a community-based cancer support group and a church-based cancer support group. Recruitment sessions were held at regular meetings of these organizations. Accrual took 2 months. In separate sessions, each male partner and each survivor completed a demographic survey and a questionnaire and were interviewed. Additionally, the couples were asked to participate in a communications activity (Adinkra). This activity was not done to fulfill any part of the study purpose and was not included in the data analysis; rather, it was done to assess its potential use as an intervention to promote dialogue between African American partners about the experience of breast cancer. ^ The questionnaire was analyzed on the basis of a coding schema and the interview responses were analyzed on the principles of hermeneutic phenomenology. In both cases, the instruments were used to determine whether the partner's coping skills reflected a compassionate attitude (positive response) versus an apathetic attitude (negative response) and whether his adaptation skills reflected supportive behaviors (the positive response) versus nonsupportive behaviors (the negative response). Overall, the women's responses showed that they perceived of their partners as being compassionate, yet nonsupportive, and the partner's perceived of themselves likewise. Only half of the women said that their partners' coping and adaptation abilities enabled them to relinquish traditional concepts of control and focus on their own well-being. ^ The themes that emerged indicate that African American men's attitudes and behaviors regarding his female partner's diagnosis of breast cancer and his ability to cope and willingness to adapt are influenced by their ritualistic mantras, folk beliefs, religious teachings/spiritual values, existential ideologies, socioeconomic status, and environmental factors and by their established perceptions of what causes breast cancer, what the treatments and outcomes are, and how the disease affects the entire family, particularly him. These findings imply that a culturally specific intervention might be useful in educating African American men about breast cancer and their roles in supporting their female partners, physically and psychologically, during diagnosis, treatment, and recovery. ^

Clinical and epidemiological characteristics of breast cancer metastases

Introduction. Breast cancer is a highly variable disease, and long-term outcomes for individual patients are difficult to predict. We evaluated a retrospective cohort of early stage breast cancer (ESBC) patients based on a variety of clinical and epidemiological factors, specifically looking at the distribution of metastasis and associations with these clinical and epidemiological factors. ^ Methods. Patients were derived from the Early Stage Breast Cancer Repository (ESBCR) with a breast cancer diagnosed between 1985 and 2000. We conducted univariate and multivariate analysis of the data to evaluate associations between characteristics and occurrence of overall, visceral, and bone metastasis. Visceral metastasis was defined as lung, liver, peritoneal, lymph node (thoracic, abdominal, pelvis), and contralateral breast cancer. ^ Results. Overall, 394 (16%) patients developed a metastasis. Of these, 83% were visceral and 17% were bone. Multivariate analyses identified the following variables to be associated with metastasis: Any metastasis: age at diagnosis, stage, ER/PR status, hormone treatment, and type of surgery (1)Visceral metastasis: age at diagnosis, stage, hormone treatment, and type of surgery (2) Bone metastasis –Alcohol use, stage, and ER/PR status ^ Discussion/conclusion. ER-/PR- status has previously been found to be associated with bone metastasis, as we confirm in our cohort. We report an association between alcohol use and bone metastasis whereas previous studies find an association with recurrence. Distribution of metastases varies from previous studies. Typically, previous studies reported bone metastasis >30%, yet our study found 17%. Previous studies varied in design, and definition of visceral metastasis. Future research is needed to further elucidate prognostic factors associated with specific metastases A more thorough understanding of what might predict which ESBC patients will develop metastases can help direct future treatment. Future studies of this nature could include the Perou intrinsic subtypes, biomarkers like Ki-67, and genetic analyses such as Oncotype DX or MammaPrint.^

Bayesian and survival model for tumor relapse status and disease-specific survival, with applications to breast cancer

Breast cancer is the most common non-skin cancer and the second leading cause of cancer-related death in women in the United States. Studies on ipsilateral breast tumor relapse (IBTR) status and disease-specific survival will help guide clinic treatment and predict patient prognosis.^ After breast conservation therapy, patients with breast cancer may experience breast tumor relapse. This relapse is classified into two distinct types: true local recurrence (TR) and new ipsilateral primary tumor (NP). However, the methods used to classify the relapse types are imperfect and are prone to misclassification. In addition, some observed survival data (e.g., time to relapse and time from relapse to death)are strongly correlated with relapse types. The first part of this dissertation presents a Bayesian approach to (1) modeling the potentially misclassified relapse status and the correlated survival information, (2) estimating the sensitivity and specificity of the diagnostic methods, and (3) quantify the covariate effects on event probabilities. A shared frailty was used to account for the within-subject correlation between survival times. The inference was conducted using a Bayesian framework via Markov Chain Monte Carlo simulation implemented in softwareWinBUGS. Simulation was used to validate the Bayesian method and assess its frequentist properties. The new model has two important innovations: (1) it utilizes the additional survival times correlated with the relapse status to improve the parameter estimation, and (2) it provides tools to address the correlation between the two diagnostic methods conditional to the true relapse types.^ Prediction of patients at highest risk for IBTR after local excision of ductal carcinoma in situ (DCIS) remains a clinical concern. The goals of the second part of this dissertation were to evaluate a published nomogram from Memorial Sloan-Kettering Cancer Center, to determine the risk of IBTR in patients with DCIS treated with local excision, and to determine whether there is a subset of patients at low risk of IBTR. Patients who had undergone local excision from 1990 through 2007 at MD Anderson Cancer Center with a final diagnosis of DCIS (n=794) were included in this part. Clinicopathologic factors and the performance of the Memorial Sloan-Kettering Cancer Center nomogram for prediction of IBTR were assessed for 734 patients with complete data. Nomogram for prediction of 5- and 10-year IBTR probabilities were found to demonstrate imperfect calibration and discrimination, with an area under the receiver operating characteristic curve of .63 and a concordance index of .63. In conclusion, predictive models for IBTR in DCIS patients treated with local excision are imperfect. Our current ability to accurately predict recurrence based on clinical parameters is limited.^ The American Joint Committee on Cancer (AJCC) staging of breast cancer is widely used to determine prognosis, yet survival within each AJCC stage shows wide variation and remains unpredictable. For the third part of this dissertation, biologic markers were hypothesized to be responsible for some of this variation, and the addition of biologic markers to current AJCC staging were examined for possibly provide improved prognostication. The initial cohort included patients treated with surgery as first intervention at MDACC from 1997 to 2006. Cox proportional hazards models were used to create prognostic scoring systems. AJCC pathologic staging parameters and biologic tumor markers were investigated to devise the scoring systems. Surveillance Epidemiology and End Results (SEER) data was used as the external cohort to validate the scoring systems. Binary indicators for pathologic stage (PS), estrogen receptor status (E), and tumor grade (G) were summed to create PS+EG scoring systems devised to predict 5-year patient outcomes. These scoring systems facilitated separation of the study population into more refined subgroups than the current AJCC staging system. The ability of the PS+EG score to stratify outcomes was confirmed in both internal and external validation cohorts. The current study proposes and validates a new staging system by incorporating tumor grade and ER status into current AJCC staging. We recommend that biologic markers be incorporating into revised versions of the AJCC staging system for patients receiving surgery as the first intervention.^ Chapter 1 focuses on developing a Bayesian method to solve misclassified relapse status and application to breast cancer data. Chapter 2 focuses on evaluation of a breast cancer nomogram for predicting risk of IBTR in patients with DCIS after local excision gives the statement of the problem in the clinical research. Chapter 3 focuses on validation of a novel staging system for disease-specific survival in patients with breast cancer treated with surgery as the first intervention. ^

Trastuzumab use and risk of congestive heart failure in older breast cancer patients

Trastuzumab is a humanized-monoclonal antibody, developed specifically for HER2-neu over-expressed breast cancer patients. Although highly effective and well tolerated, it was reported associated with Congestive Heart Failure (CHF) in clinical trial settings (up to 27%). This leaves a gap where, Trastuzumab-related CHF rate in general population, especially older breast cancer patients with long term treatment of Trastuzumab remains unknown. This thesis examined the rates and risk factors associated with Trastuzumab-related CHF in a large population of older breast cancer patients. A retrospective cohort study using the existing Surveillance, Epidemiology and End Results (SEER) and Medicare linked de-identified database was performed. Breast cancer patients ≥ 66 years old, stage I-IV, diagnosed in 1998-2007, fully covered by Medicare but no HMO within 1-year before and after first diagnosis month, received 1st chemotherapy no earlier than 30 days prior to diagnosis were selected as study cohort. The primary outcome of this study is a diagnosis of CHF after starting chemotherapy but none CHF claims on or before cancer diagnosis date. ICD-9 and HCPCS codes were used to pool the claims for Trastuzumab use, chemotherapy, comorbidities and CHF claims. Statistical analysis including comparison of characteristics, Kaplan-Meier survival estimates of CHF rates for long term follow up, and Multivariable Cox regression model using Trastuzumab as a time-dependent variable were performed. Out of 17,684 selected cohort, 2,037 (12%) received Trastuzumab. Among them, 35% (714 out of 2037) were diagnosed with CHF, compared to 31% (4784 of 15647) of CHF rate in other chemotherapy recipients (p<.0001). After 10 years of follow-up, 65% of Trastuzumab users developed CHF, compared to 47% in their counterparts. After adjusting for patient demographic, tumor and clinical characteristics, older breast cancer patients who used Trastuzumab showed a significantly higher risk in developing CHF than other chemotherapy recipients (HR 1.69, 95% CI 1.54 - 1.85). And this risk is increased along with the increment of age (p-value < .0001). Among Trastuzumab users, these covariates also significantly increased the risk of CHF: older age, stage IV, Non-Hispanic black race, unmarried, comorbidities, Anthracyclin use, Taxane use, and lower educational level. It is concluded that, Trastuzumab users in older breast cancer patients had 69% higher risk in developing CHF than non-Trastuzumab users, much higher than the 27% increase reported in younger clinical trial patients. Older age, Non-Hispanic black race, unmarried, comorbidity, combined use with Anthracycline or Taxane also significantly increase the risk of CHF development in older patients treated with Trastuzumab. ^

ROLE OF MIR-19A RELEASED BY INFLAMMATORY BREAST CANCER CELLS IN THE REGULATION OF DENDRITIC CELL FUNCTIONS: IN VITRO MODEL OF CROSSTALK IN THE TUMOR MICROENVIRONMENT OF INFLAMMATORY BREAST CANCER

Inflammatory breast cancer (IBC) is a rare but very aggressive form of locally advanced breast cancer (1-6% of total breast cancer patients in United States), with a 5-year overall survival rate of only 40.5%, compared with 85% of the non-IBC patients. So far, a unique molecular signature for IBC able to explain the dramatic differences in the tumor biology between IBC and non-IBC has not been identified. As immune cells in the tumor microenvironment plays an important role in regulating tumor progression, we hypothesized that tumor-associated dendritic cells (TADC) may be responsible for regulating the development of the aggressive characteristics of IBC. MiRNAs can be released into the extracellular space and mediate the intercellular communication by regulating target gene expression beyond their cells of origin. We hypothesized that miRNAs released by IBC cells can induce an increased activation status, secretion of pro-inflammatory cytokines and migration ability of TADC. In an in vitro model of IBC tumor microenvironment, we found that the co-cultured of the IBC cell line SUM-149 with immature dendritic cells (iDCSUM-149) induced a higher degree of activation and maturation of iDCSUM-149 upon stimulation with lipopolysaccharide (LPS) compared with iDCs co-cultured with the non-IBC cell line SUM-159 (iDCSUM-159), resulting in: increased expression of the costimulatory and activation markers; higher production of pro-inflammatory cytokines (TNF-a, IL-6); and 3) higher migratory ability. These differences were due to the exosome-mediated transfer of miR-19a and miR-146a from SUM-149 and SUM-159, respectively, to iDCs, causing the downregulation of the miR-19a target genes PTEN, SOCS-1 and the miR-146a target genes IRAK1, TRAF6. PTEN, SOCS-1 and IRAK1, TRAF6 are important negative and positive regulator of cytokine- and TLR-mediated activation/maturation signaling pathway in DCs. Increased levels of IL-6 induced the upregulation of miR-19a synthesis in SUM-149 cells that was associated with the induction of CD44+CD24-ALDH1+ cancer stem cells (CSCs) with epithelial-to-mesenchymal transition (EMT) characteristics. In conclusion, in IBC tumor microenvironment IL-6/miR-19a axis can represent a self-sustaining loop able to maintain a pro-inflammatory status of DCs, leading to the development of tumor cells with high metastatic potential (EMT CSCs) responsible of the poor prognosis in IBC patients.

The role of erbB2 receptor in human breast cancer metastasis

Overexpression of c-erbB-2 gene-encoded p185 has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of c-erbB-2 can enhance metastatic potential of human breast cancer cells, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the c-erbB-2 gene transfected 435.eB cells. In vivo experimental metastasis assays demonstrated that mice injected erbB2-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in metastatic potential in vivo were accompanied by increased invasiveness in vitro . The transfectants and the parental cells all had similar growth rates and transformation potential. These findings suggest that c- erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities. ^ Homophilic adhesion may affect invasive and metastatic potential of tumor cells. We found that Heregulin-β1 (HRG-β1), a growth factor that activates receptor kinases erbB3 and erbB4, can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-β1-increased cell aggregation, we observed that HRG-β1 increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using different kinase inhibitors, we found that the HRG-β1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-β1-activated PI3K is required for enhancing breast cancer cell aggregation. These results have provided one mechanism by which HRG-β1-activated signaling of erbB receptors may affect invasive/metastatic properties of breast cancer cells. ^ To identify the structural motifs within the erbB2 receptor that are required for erbB2 increased metastatic potential in breast cancer cells, we injected different forms of mutated erbB2 expressing MDA-MB-435 cell line transfectants with or without the EGF-like domain of heregulin-β1 protein (HRG/egf) into ICR-SCID mice to test the metastatic survival rate. The results show that an intact kinase domain of erbB2 receptor is required for erbB2 enhanced metastatic potential in these cells. The C-terminal tyrosine 1248 residue of erbB2 may also play a role in enhancing metastatic potential. Moreover, the results suggest that HRG/egf promote the metastatic potential of human breast cancer cells in vivo. ^

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^

Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary myoepithelial cells

A serpin was identified in normal mammary gland by differential cDNA sequencing. In situ hybridization has detected this serpin exclusively in the myoepithelial cells on the normal and noninvasive mammary epithelial side of the basement membrane and thus was named myoepithelium-derived serine proteinase inhibitor (MEPI). No MEPI expression was detected in the malignant breast carcinomas. MEPI encodes a 405-aa precursor, including an 18-residue secretion signal with a calculated molecular mass of 46 kDa. The predicted sequence of the new protein shares 33% sequence identity and 58% sequence similarity to plasminogen activator inhibitor (PAI)-1 and PAI-2. To determine whether MEPI can modulate the in vivo growth and progression of human breast cancers, we transfected a full-length MEPI cDNA into human breast cancer cells and studied the orthotopic growth of MEPI-transfected vs. control clones in the mammary fat pad of athymic nude mice. Overexpression of MEPI inhibited the invasion of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, the primary tumor volumes, axillary lymph node metastasis, and lung metastasis were significantly inhibited in MEPI-transfected clones as compared with controls. The expression of MEPI in myoepithelial cells may prevent breast cancer malignant progression leading to metastasis.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.