Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

Axillary lymph node dissection versus no dissection in patients with T1N0 breast cancer: a randomized clinical trial (INT09/98).

BACKGROUND: Although axillary surgery is still considered to be a fundamental part of the management of early breast cancer, it may no longer be necessary either as treatment or as a guide to adjuvant treatment. The authors conducted a single-center randomized trial (INT09/98) to determine the impact of avoiding axillary surgery in patients with T1N0 breast cancer and planning chemotherapy based on biological factors of the primary tumor on long-term disease control. METHODS: From June 1998 to June 2003, 565 patients aged 30 years to 65 years with T1N0 breast cancer were randomized to either quadrantectomy with (QUAD) or without (QU) axillary lymph node dissection; a total of 517 patients finally were evaluated. All patients received radiotherapy to the residual breast only. Chemotherapy for patients in the QUAD treatment arm was determined based on lymph node status, estrogen receptor status, and tumor grade. Chemotherapy for patients in the QU treatment arm was based on estrogen receptor status, tumor grade, and human epidermal growth factor receptor 2 and laminin receptor status. Overall survival (OS) was the primary endpoint. Disease-free survival (DFS) and rate and time of axillary lymph node recurrence in the QU treatment arm were the secondary endpoints. RESULTS: After a median follow-up of >10 years, the estimated adjusted hazards ratio of the QUAD versus QU treatment arms for OS was 1.09 (95% confidence interval, 0.59-2.00; P = .783) and was 1.04 (95% confidence interval, 0.56-1.94; P = .898) for DFS. Of the 245 patients in the QU treatment arm, 22 (9.0%) experienced axillary lymph node recurrence. The median time to axillary lymph node recurrence from breast surgery was 30.0 months (interquartile range, 24.2 months-73.4 months). CONCLUSIONS: Patients with T1N0 breast cancer did not appear to benefit in terms of DFS and OS from immediate axillary lymph node dissection in the current randomized trial. The biological characteristics of the primary tumor appear adequate for guiding adjuvant treatment.

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy.

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

Increased overall survival independent of RECIST response in metastatic breast cancer patients continuing trastuzumab treatment: evidence from a retrospective study.

Recent studies have reported the potential clinical utility for metastatic breast cancer (MBC) patients of continuing trastuzumab beyond progression. Based on those results, here the authors have examined the benefits of trastuzumab-continuation by specifically evaluating RECIST responses upon first line trastuzumab-treatment as a potential predictive marker for therapeutic effect of trastuzumab-continuation beyond metastatic disease progression. The authors carried out a retrospective analysis of 272 HER2 positive MBC patients under trastuzumab treatment at 22 different oncology Italian centers during the years of 2000 and 2001 who progressed under first line trastuzumab-treatment. The primary end point of the study was the survival from the date of first documented progression upon first line trastuzumab treatment of disease. Data analysis involved the use of matching on propensity score to balance variables between treated and untreated subjects and to reduce bias. Of the 272 HER2-positive MBC patients, 154 (56.6%) continued treatment. 79 (51.3%) of those 154 patients showed responses based on RECIST criteria during first-line trastuzumab-treatment. Of the 118 patients that suspended trastuzumab, RECIST responses had been observed in 44 (37.3%). Cox proportional hazards analysis of progressed patients, matched using propensity score, showed that discontinuation of trastuzumab at metastatic disease progression was a risk factor for significantly reduced overall survival in both responder (HR = 2.23; 95% CI = 1.03-4.82) and non-responder groups (HR = 3.53, 95% CI = 1.73-7.21), with no significant differences in the two estimated HRs (P-value of the likelihood-ratio test = 0.690). Continued trastuzumab treatment after disease progression has clinically and statistically significant effects in both RECIST responder and non-responder MBC patients.

Shed HER2 extracellular domain in HER2-mediated tumor growth and in trastuzumab susceptibility.

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.

Critical role of TLR9 in acute graft-versus-host disease.

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9(-/-)) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4(-/-)) mice. The reduced mortality and morbidity in TLR9(-/-) mice related with reduced stimulatory activity of TLR9(-/-) spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9(+/+) into TLR9(-/-) and TLR9(-/-) into TLR9(+/+) chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9(+/+) cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.

Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab.

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.

Sex hormone levels, breast cancer risk, and cancer receptor status in postmenopausal women: the ORDET cohort.

BACKGROUND: Endogenous sex hormone levels have been associated with increased breast cancer risk in postmenopausal women in several prospective studies. However, it remains unclear to what extent serum hormone-breast cancer associations differ with receptor status. METHODS: Associations between serum sex hormone levels and breast cancer risk were assessed in a nested case-control study on postmenopausal women of the ORDET cohort. After a median follow-up of 13.5 years, 165 women developed breast cancer. Relative risks of developing breast cancer were estimated by conditional logistic regression. RESULTS: Total and free testosterone levels were directly associated with breast cancer risk [relative risk, 3.28 (95% confidence interval, 1.93-5.55) and 2.86 (95% confidence interval, 1.66-4.94), respectively, for highest versus lowest quartile]. When relations between hormone level and risk of breast cancer expressing various receptor combinations were assessed, high total testosterone was significantly associated with increased risk of estrogen receptor-positive cancers, irrespective of progesterone receptor status. High total testosterone was also associated with increased risk of both human epidermal growth factor receptor 2 (HER2)-negative (HER2(-)) and HER2(+) cancers. High estradiol tended to be associated with increased risk of HER2(-) cancer and inversely associated with HER2(+) cancer, with significant (P = 0.027) heterogeneity between HER2(+) and HER2(-) cancers. However, there were relatively few HER2(+) cases. CONCLUSIONS: This study provides further evidence that high levels of circulating testosterone increase the risk of developing breast cancer in postmenopausal women. The cancers that develop are mainly estrogen receptor positive. Although HER2(+) and HER2(-) breast cancers were both associated with high total testosterone, they showed opposing associations with estrogen.

Eradication of ovarian tumor xenografts by locoregional administration of targeted immunotherapy.

PURPOSE: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) are potent activators of innate and adaptive immunity. Recognition of CpG-ODN is mediated by Toll-like receptor 9 expressed by immune cells, endothelial and epithelial cells, and fibroblasts. We examined the antitumor effect of CpG-ODN and the role of administration route on human ovarian cancers growing in the peritoneal cavity of nude mice. EXPERIMENTAL DESIGN: Mice implanted i.p. with human ovarian carcinoma cells were treated i.p., s.c., or i.v. and assessed for survival and tumor-free incidence. Peritoneal washings were analyzed for keratinocyte chemokine production and for functional and phenotypic profiles as indicators of the cell types involved in mediating the antitumor effects. RESULTS: IGROV-1-bearing mice treated i.p. survived significantly longer than those treated i.v. or s.c. (P=0.0005), and nearly half of them (8 of 17) were tumor-free by the end of the experiment, a rate never achieved using a variety of chemotherapeutic drugs. High rates of tumor-free mice were observed in three other ovarian tumor xenografts treated i.p. Compared with peritoneal washings of mice treated s.c. or i.v., those from mice treated i.p. showed the highest level of serum and tissue keratinocyte chemokine, the highest number of natural killer cells and neutrophils, and the highest antiproliferative activity in vitro. CONCLUSIONS: The superior antitumor effect obtained by locoregional administration of CpG-ODN in i.p. tumor-bearing mice with a limited adaptive immune response points to the importance of innate effector cells amplification at the site of tumor growth and suggests the promise of i.p. CpG-ODN in clinical trials for ovarian cancer.

Two distinct local relapse subtypes in invasive breast cancer: effect on their prognostic impact.

PURPOSE: Local relapse (LR) remains an important concern in breast cancer surveillance. Given the unique opportunity to study local recurrences in the breast in the absence of irradiation, we tested the possibility of identifying a particular tumor feature that might account for LR. EXPERIMENTAL DESIGN: Archival specimens from 235 patients belonging to the control arm (no radiotherapy) of the Milan 3 Trial were retrieved and included in this study. H&E-stained histologic slides were reviewed for diagnostic reassessment. A panel of biological variables was assessed by immunohistochemical/cytochemical staining. RESULTS: Onset of LR was significantly linked only to patient's age, with a hazard ratio (HR) to relapse reduced by 60% in patients >50 years of age (P = 0.002). Univariate and multivariate Cox analyses in women 50 years of age, lymph node and estrogen receptor status were significantly predictive of LR (HR, 3.34; P = 0.0113 and HR, 0.39; P = 0.0424, respectively). Multivariate analysis indicated that apart from lymph node positivity (HR, 3.48; P = 0.0120), none of the variables associated with LR in univariate analysis displayed an independent LR-predictive power. LR had a strong prognostic impact on distant metastasis in patients >50 years of age, whereas in younger women, LR did not affect the risk of metastasis. CONCLUSIONS: These results support the notion that LR arises from two distinct mechanisms, one that is more frequent in young patients, associated with host characteristics, and is not linked with prognosis, and another that is less frequent, but is associated with tumor aggressiveness which represents a peculiar and distinct marker of breast tumor malignancy.

Protein kinase Calpha determines HER2 fate in breast carcinoma cells with HER2 protein overexpression without gene amplification.

In some HER2-positive breast tumors, cell surface overexpression of HER2 is not associated with gene amplification but may instead rest in altered gene transcription, half-life, or recycling of the oncoprotein. Here, we show that HER2 overexpression in HER2 2+ carcinomas is associated with neither an increase in gene transcription nor a deregulation in the ubiquitin-dependent pathways, but instead seems to be regulated by protein kinase Calpha (PKCalpha) activity. The stimulation of PKCalpha up-regulated HER2 expression, whereas PKCalpha inhibition by pharmacologic treatments and PKCalpha-specific small interfering RNA led to a dramatic down-regulation of HER2 levels only in breast cancer cells HER2 2+. Consistent with the in vitro data, our biochemical analysis of HER2 2+ human primary breast specimens revealed significantly higher levels of phosphorylated PKCalpha compared with HER2-negative tumors. Inhibition of HER2 activation by the tyrosine kinase inhibitor lapatinib led to decreased levels of PKCalpha phosphorylation, clearly indicating a cross-talk between PKCalpha and HER2 molecules. These data suggest that HER2 overexpression in HER2 2+ carcinomas is due to an accumulation of the recycled oncoprotein to the cell surface induced by activated PKCalpha.

Regulation of breast cancer response to chemotherapy by fibulin-1.

Doxorubicin treatment was found to augment the expression of the extracellular matrix (ECM) protein fibulin-1 in cultured human breast cancer cell lines and in MDA-MB-361 tumors grown in athymic mice. Doxorubicin was also found to augment tumor expression of the fibulin-1-binding proteins fibronectin and laminin-1. Growth of breast cancer cell lines on Matrigel, an ECM extract containing fibulin-1 and laminin-1, resulted in lower levels of doxorubicin-induced apoptosis as compared with controls. Moreover, tumors formed by injection of athymic mice with MDA-MB-361 cells mixed with Matrigel were significantly more doxorubicin resistant and displayed lower levels of apoptosis compared with those that formed in the absence of Matrigel. Monoclonal antibodies against fibulin-1 reversed Matrigel-dependent doxorubicin resistance. Furthermore, small interfering RNA-mediated suppression of fibulin-1 expression in breast cancer cells resulted in a 10-fold increase in doxorubicin sensitivity as compared with control cells. Together, these findings point to a role for fibulin-1 in breast cancer chemoresistance.

MicroRNA signatures in human ovarian cancer.

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide and, despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. Unfortunately, still relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The relatively recent discovery of microRNAs (miRNA), a class of small noncoding RNAs targeting multiple mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Multiple studies involving various types of human cancers proved that miRNAs have a causal role in tumorigenesis. Here we show that, in comparison to normal ovary, miRNAs are aberrantly expressed in human ovarian cancer. The overall miRNA expression could clearly separate normal versus cancer tissues. The most significantly overexpressed miRNAs were miR-200a, miR-141, miR-200c, and miR-200b, whereas miR-199a, miR-140, miR-145, and miR-125b1 were among the most down-modulated miRNAs. We could also identify miRNAs whose expression was correlated with specific ovarian cancer biopathologic features, such as histotype, lymphovascular and organ invasion, and involvement of ovarian surface. Moreover, the levels of miR-21, miR-203, and miR-205, up-modulated in ovarian carcinomas compared with normal tissues, were significantly increased after 5-aza-2'-deoxycytidine demethylating treatment of OVCAR3 cells, suggesting that the DNA hypomethylation could be the mechanism responsible for their overexpression. Our results indicate that miRNAs might play a role in the pathogenesis of human EOC and identify altered miRNA gene methylation as a possible epigenetic mechanism involved in their aberrant expression.

Matured human monocyte-derived dendritic cells (MoDCs) induce expansion of CD4+CD25+FOXP3+ T cells lacking regulatory properties.

Naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs) play a key role as suppressors in immune mechanisms that protect against self-destruction. The forkhead box p3 transcription factor (FOXP3) has a central role in the development of nTregs. We show here that co-culture of naïve T cells with flagellin-exposed monocyte-derived dendritic cells (MoDCs) generates CD4(+)CD25(+)FOXP3(+) T cells that transiently express FOXP3 together with CD25 but do not suppress proliferation of CD4(+)CD25(-) T cells. Moreover, purified CD4(+)CD25(+)FOXP3(+) T cells reveal a different proliferation and cytokine production profile from that of nTregs. These data indicate that in the presence of ongoing immune responses a T cell antigenic phenotype superimposable of that of nTregs does not necessarily predict suppressive function and that FOXP3 in humans is not sufficient for development and function of regulatory T cells.

Radiation effects on development of HER2-positive breast carcinomas.

PURPOSE: Neither hormone-related nor genetics risk factors have been associated with the development of highly proliferative HER2-positive breast carcinomas. Because the majority of HER2-positive tumors present the amplification of the oncogene, we asked whether genomic instability triggered by irradiation might be involved in the induction of HER2-overexpressing breast carcinomas. EXPERIMENTAL DESIGN: Sixty-six infiltrating breast carcinomas from patients treated with radiation therapy for Hodgkin's lymphoma or other pediatric solid tumors and a control series of 61 consecutive sporadic breast tumors were analyzed by immunohistochemistry for HER2 expression with HercepTest. A panel of antibodies against estrogen receptor, progesterone receptor, c-kit, cytokeratin 5/6, p53, and ki67 antigen was also used to identify differentiation subsets and molecular characteristics of the analyzed breast carcinomas. RESULTS: Although no differences between the two tumor series were found with respect to HER2 expression scored 2+ and 3+, the percentage of 3+ HER2-positive tumors was significantly higher in patients irradiated during breast maturation compared with patients irradiated after breast maturation (35.3% versus 12.5%, P = 0.046). In the latter group, 52.5% of the breast carcinomas showed basal-like differentiation (estrogen receptor, progesterone receptor, and HER2 negative) versus only 5.9% in the group irradiated during breast development (P < 0.0001). Analysis adjusted for age confirmed the significant increase in basal-like tumor development in patients irradiated within 4 years of menarche, but also showed that the differences between patients irradiated before and after puberty in HER2 3+ tumor frequencies are due to age-related differences in HER2 3+ tumor onset. CONCLUSION: Together, our data indicate that the development of HER2-positive tumors correlates with timing rather than type of carcinogenic hits and provide clear evidence that radiation is a risk factor for breast carcinomas showing basal-like differentiation.

Cross-talk between toll-like receptors 5 and 9 on activation of human immune responses.

The recognition of pathogen-associated molecular patterns by TLRs triggers the activation of innate and adaptive immune responses. Flagellin, the agonist of TLR5, is expressed by prokaryotes and eukaryotes, and DNA sequences containing unmethylated CpG dinucleotides, agonists of TLR9, are present essentially in prokaryotes. To test the potential modulating effects of simultaneous activation of different TLRs on the immune response, we compared the outcomes in different immune cell compartments induced by triggering TLR5 and TLR9 individually and in combination. PBMCs, monocytes, and monocyte-derived DC (MoDC) secreted high levels of IL-10 in response to flagellin, whereas oligodeoxynucleotides (ODN) containing the CpG sequence (CpG-ODN), synthetic ligands of TLR9, did not induce IL-10 secretion in any of the three cell types but synergized with flagellin in this induction. In contrast, PBMC production of IFN-alpha induced by CpG-ODN was strongly inhibited by flagellin. Conversely, CpG-ODN did not enhance the up-regulation of activation markers in MoDC induced to mature in the presence of flagellin. Flagellin-matured, but not CpG-ODN-matured, MoDC stimulated the expansion of allogeneic CD4+CD25+ T cells, and the extent of expansion induced by MoDC, matured in the presence of flagellin and CpG-ODN, was similar to that induced by flagellin-matured MoDC. Moreover, flagellin and CpG-ODN differentially affected NK-mediated cytotoxicity, and flagellin completely abrogated the NK-mediated immune response induced by CpG-ODN stimulation. Together, these results suggest that flagellin inhibits the TLR9-induced cell activation and cytokine production, which favor Th1-type immune responses, possibly because the signals evoked by flagellin to indicate the presence of extracellular pathogens must favor a Th2-polarized response. Thus, TLR5 and TLR9, alerted by the presence of microorganisms, influence each other to mount the more efficient and appropriate immune response to contain the infection of a specific pathogen.

Influence of antibiotic treatment on breast carcinoma development in proto-neu transgenic mice.

The effect of prolonged antibiotic treatments on tumor development was evaluated in proto-neu transgenic mice, which spontaneously develop mammary carcinomas. Virgin transgenic mice were treated with metronidazole/ciprofloxacin or gentamicin through the drinking water. The hazard ratio [HR; 95% confidence interval (95% CI)] of breast cancer occurrence in metronidazole/ciprofloxacin-treated mice was more than triple that for controls [3.11 (1.13-8.53); P = 0.028], whereas only a slight increase in HR (95% CI) was observed in gentamicin-treated mice [1.39 (0.56-3.47); P = 0.481]. Tumor growth rate in gentamicin-treated mice was significantly faster than in untreated control mice (P = 0.043). Moreover, mammary glands from mice treated with either antibiotic regimen showed increased lobulization, with more numerous and more developed terminal ductal lobular units than in controls. These results indicate that prolonged exposure to relevant doses of antibiotics affects the mammary glands in this particular model of HER-2/neu transgenic mice; further studies to understand the precise mechanism by which antibiotic treatments influence mammary gland differentiation are critical.

Relationship between p53 and p27 expression following HER2 signaling.

HER2, frequently associated with low p27 expression in breast tumors, when activated has been found to upmodulate p53 in tumor cells. The aim of this work was to investigate the role of p53 in the connection between HER2 and p27. Fifty-two breast tumor specimens, characterized for p53 mutations, were analyzed immunohistochemically (IHC) for HER2, p53 and p27 expression. p27, inversely associated with HER2, was found in 29% of tumors with IHC-negative mutated p53 versus 93% of tumors with accumulation of p53 protein and 59% with wild-type p53 (p=0.001), indicating a direct association between p53 and p27 expression. HER2-overexpressing cell lines carrying wild-type or null p53 protein, and treated with heregulin beta1 (HRG), were analyzed for expression and subcellular localization of p53 and p27. In HER2-overexpressing cells stimulated with HRG, p27 protein expression increased in parallel with p53 with no corresponding increase in p27 transcript. No p27 increase was observed in p53-null cells. Transfection with wild-type p53 restored p27 upmodulation in HRG-stimulated cells, indicating a crucial role of p53 in determining p27 upmodulation following HER2 activation. Together, our data demonstrate the crucial role of p53 in determining p27 upmodulation following HER2 activation. This could have implications in the response to Transtuzumab therapy.

Inhibition of mammary carcinoma development in HER-2/neu transgenic mice through induction of autoimmunity by xenogeneic DNA vaccination.

Plasmid DNA vectors encoding the full-length (VR1012/HER-2-FL) or only the extracellular and transmembrane domains (VR1012/HER-2-ECD-TM) of human (h) HER-2/neu proto-oncogene were used to vaccinate HER-2/neu transgenic mice (N202) engineered to overexpress the rat (r) neu proto-oncogene product (r-p185(neu)). Both the full-length and the deleted vaccines were significantly (P = 0.0001 and P = 0.06, respectively) more active than the empty vector (VR1012/EV) in preventing and delaying HER-2/neu-driven mammary carcinogenesis. A low-level intratumoral infiltrate of dendritic cells, macrophages, CD8 T cells and polymorphonuclear granulocytes in association with low-level cytokine production was observed, which was not detected in tumors from control mice. Morphologic analyses showed that vaccination with VR1012/HER-2-FL or ECD-TM also efficiently hampered the development of terminal ductal lobular units (TDLU). Analyses of sera from vaccinated mice revealed high titers of antihuman HER-2/neu antibodies, which correlated with the delayed time of tumor onset (P = 0.002). These antibodies did not cross-react with r-p185(neu). Nontransgenic mice treated with the vaccines produced autoreactive antibodies targeting mouse (m)-p185(neu) and showed impaired function of the lactating mammary gland and accelerated involution of the gland after weaning. Together, these data indicate that xenogeneic DNA immunization breaks tolerance against the endogenous m-p185(neu), impairing the development of mammary TDLU in which m-p185(neu) expression is concentrated. The reduction in the number of TDLU decreases the number of glandular structures available for r-p185(neu)-dependent mammary carcinogenesis, resulting in a significant inhibition of mammary carcinoma development.

Therapeutic synergism of gemcitabine and CpG-oligodeoxynucleotides in an orthotopic human pancreatic carcinoma xenograft.

CpG-oligodeoxynucleotides (CpG-ODN) exhibit potent immunostimulatory activity by binding with Toll-like receptor 9 (TLR9). Based on the finding that TLR9 is highly expressed and functional in pancreatic tissue, we evaluated the antitumor effects of chemotherapy combined with CpG-ODNs in the orthotopic mouse model of a human pancreatic tumor xenograft. Chemotherapy consisted of the maximum tolerated dose of gemcitabine (i.v., 100 mg/kg, q3dx4). CpG-ODNs were delivered (i.p., 20 microg/mouse), weekly, after the end of chemotherapy. CpG-ODNs alone had little effect on tumor growth, whereas gemcitabine alone significantly delayed the median time of disease onset (palpable i.p. tumor) and of bulky disease development (extensive peritoneal tumor burden), but did not enhance survival time. When the gemcitabine regimen was followed by administration of the immunostimulator, development of bulky disease was delayed, survival time was significantly improved (median survival time, 106 days; P < 0.02 versus gemcitabine-treated mice). Autoptic examination showed that tumor spread in the peritoneal cavity was reduced to a greater extent than with gemcitabine alone. All treatment regimens were well-tolerated. The use of nude mice excluded a T cell-mediated immune response, whereas the high pancreatic expression of TLR9 might have contributed to the tumor response. The clear improvement of survival observed in an orthotopic murine model of human pancreatic cancer by the combined use of CpG-ODNs with chemotherapy suggests the promise of this therapeutic regimen in the clinical setting.