Her2/neu signaling blockade improves tumor oxygenation in a multifactorial fashion in Her2/neu+ tumors

Purpose  Tumor hypoxia reduces the efficacy of radiation and chemotherapy as well as altering gene expression that promotes cell survival and metastasis. The growth factor receptor, Her2/neu, is overexpressed in 25–30% of breast tumors. Tumors that are Her2⁺ may have an altered state of oxygenation, relative to Her2⁻ tumors, due to differences in tumor growth rate and angiogenesis. Methods  Her2 blockade was accomplished using an antibody to the receptor (trastuzumab; Herceptin). This study examined the effects of Her2 blockade on tumor angiogenesis, vascular architecture, and hypoxia in Her2⁺ and Her2⁻ MCF7 xenograft tumors. Results  Treatment with trastuzumab in Her2⁺ tumors significantly improved tumor oxygenation, increased microvessel density, and improved vascular architecture compared with the control-treated Her2⁺ tumors. The Her2⁺ xenografts treated with trastuzumab also demonstrated decreased proliferation indices when compared with control-treated xenografts. These results indicate that Her2 blockade can improve tumor oxygenation by decreasing oxygen consumption (reducing tumor cell proliferation and inducing necrosis) and increasing oxygen delivery (vascular density and architecture). Conclusions  These results support the use of trastuzumab as an adjunct in the treatment of breast tumors with chemotherapy or radiotherapy, as improvements in tumor oxygenation should translate into improved treatment response.     

Cdk2-Null Mice Are Resistant to ErbB-2-Induced Mammary Tumorigenesis

The concept of targeting G₁ cyclin-dependent kinases (CDKs) in breast cancer treatments is supported by the fact that the genetic ablation of Cdk4 had minimal impacts on normal cell proliferation in majority of cell types, resulting in near-normal mouse development, whereas such loss of Cdk4 completely abrogated ErbB-2/neu-induced mammary tumorigenesis in mice. In most human breast cancer tissues, another G₁-regulatory CDK, CDK2, is also hyperactivated by various mechanisms and is believed to be an important therapeutic target. In this report, we provide genetic evidence that CDK2 is essential for proliferation and oncogenesis of murine mammary epithelial cells. We observed that 87% of Cdk2-null mice were protected from ErbB-2-induced mammary tumorigenesis. Mouse embryonic fibroblasts isolated from Cdk2-null mouse showed resistance to various oncogene-induced transformation. Previously, we have reported that hemizygous loss of Cdc25A, the major activator of CDK2, can also protect mice from ErbB-2-induced mammary tumorigenesis [Cancer Res (2007) 67(14): 6605–11]. Thus, we propose that CDC25A-CDK2 pathway is critical for the oncogenic action of ErbB-2 in mammary epithelial cells, in a manner similar to Cyclin D1/CDK4 pathway.     

Murine pneumotropic virus chimeric Her2/neu virus-like particles as prophylactic and therapeutic vaccines against Her2/neu expressing tumors

Virus-like particles (VLPs) have increasingly attracted attention as DNA-free and safe antigen carriers in tumor immunotherapy, requiring only minute amounts of antigens. Previously, we have immunized with murine polyomavirus (MPyV) VLPs carrying human Her2/neu and prevented the outgrowth of a human Her2/neu expressing tumor in a transplantable tumor model as well as outgrowth of spontaneous rat Her2/neu carcinomas in BALB-neuT mice. Here, we examine if prophylactic and therapeutic protection could be obtained with murine pneumotropic virus (MPtV) VLPs, and study the cross-reactivity between human and rat Her2/neu. VLPs from MPyV and MPtV carrying human or rat Her2/neu were tested in two transplantable tumor models against a human Her2/neu positive (D2F2/E2) and a rat Her2/neu positive tumor cell line (TUBO). Rat Her2/neu-VLPs were also tested in BALB-neuT mice. Her2/neu-MPtVLPs were as efficient as prophylactic vaccines against D2F2/E2 and TUBO as those from MPyV. Homologous Her2/neu was better than heterologous, i.e. human Her2/neu-VLPs were better than rat Her2/neu-VLPs against D2F2/E2 and vice versa. Moreover, therapeutic immunization with human Her2/neu-VLPs together with CpG given up to 6 days after challenge protected against D2F2/E2. In BALB-neuT mice, rat Her2/neu-VLPs were less efficient than human Her2/neu-VLPs used in our previous study, implying that protection seen in that study was partly due to the use of human rather than rat Her2/neu. In conclusion, Her2/neu-MPtVLPs are effective both as prophylactic and therapeutic tumor vaccines. Homologous Her2/neu-VLPs are superior to heterologous in transplantable tumor models, while the opposite is true in BALB-neuT mice.     

Prevention of Mammary Tumorigenesis in Acatalasemic Mice by Vitamin E Supplementation

Adult male and female acatalasemic (C3H/AnLCs^bCs^b), hypocatalasemic (C3H/AnLCs^cCs^c) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E-deficient (28 animals) and vitamin E-supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E-supplemented diet was 47%, while that in mice given vitamin E-deficient diet was 82% ( P <0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.     

Cox-2 and Her2/neu co-expression in invasive bladder cancer

We examined the expression of Her2/neu and Cox-2 in bladder cancer and its relationship to clinicopathological factors, survival data and patient outcome. From 153 consecutive patients who had undergone radical cystectomy for bladder cancer, tumour tissue was analysed for Her2/neu amplification by fluorescent in situ hybridisation and for Her2/neu and Cox-2 protein expression by immunohistochemistry. Results were correlated with clinical data and survival times. Cox-2 and Her2/neu co-expression was present in 44 (33%) of 132 transitional cell carcinomas. Although this association was significant (p = 0.003), there was no significant association between Cox-2 and Her2/neu amplification status. Each marker was independent of primary tumour stage and lymph node status, as well as histological grading. However, the co-existence of Her2/neu amplification and Cox-2 expression correlated with distant metastases: of 5 Cox-2-positive samples, 2 (40%) showed Her2/neu amplification. This was only a trend, amounting to no more than borderline statistical significance (p = 0.046). Kaplan-Meier survival analysis did not demonstrate any relationship between Cox-2 or Her2/neu expression or amplification alone or in combination with respect to overall and disease-free survival. Analysing the co-expression of Cox-2 and Her2/neu status does not add any prognostic information in patients with bladder cancer. Nevertheless, combined treatment with Her2/neu and Cox-2 inhibitors may be beneficial for the subgroup of Her2/neu- and Cox-2-expressing tumours and will have to be assessed in further preclinical and clinical studies.     

Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2

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Estrogen Receptor-beta Mediates the Protective Effects of Aromatase Induction in the MMTV-Her-2/neu x Aromatase Double Transgenic Mice

Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERβ-selective antagonist THC reversed these changes. The regulation of these factors by ERβ was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERβ, suggesting that ERβ may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERβ may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERβ agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.     

Her2/neu testing in gastric cancer: evaluating the risk of sampling errors

BackgroundWe evaluated the risk of sampling errors in specimens of biopsy size, which may be caused by heterogeneous overexpression of Her2/neu in gastric cancer (GC).Patients and methodsThe study cohort comprised 454 gastrectomy patients with adenocarcinoma of the stomach or esophago-gastric junction. Tissue micro-arrays (TMAs) served as ‘biopsy procedure’ and were generated from formalin-fixed and paraffin-embedded tissue: five tissue cylinders were collected randomly from each tumor, rendering 2230 core cylinders. These were compared with 454 whole tissue sections obtained from the same paraffin blocks. Her2/neu expression and gene amplification were analyzed by immunohistochemistry and in situ hybridization. The Her2/neu status was determined according to GC scoring system by two independent observers.ResultsIn whole tissue sections, 37 (8.1%; observer 1) and 38 (8.4%; observer 2) of the GCs, and in the corresponding TMAs, 28 (6.3%; observer 1) and 28 (6.3%; observer 2) of the GCs were classified as Her2/neu-positive (kappa value 98.5% and 96.2%; P < 0001). Comparison of whole tissue sections with corresponding TMAs showed a false-negative rate of 24% and a false-positive rate of 3% for TMAs.ConclusionAssessment of the Her2/neu status in tissue biopsies carries a significant risk of sampling errors, thereby rendering patients unsuitable for treatment with trastuzumab.     

COX-2及Her2/neu在结直肠癌旁组织中表达意义

目的 检测结直肠癌和旁癌组织中的COX-2及Her2/neu的表达情况,探究COX-2及Her2/neu表达与结直肠癌临床各指标的关系,并探讨其临床意义.方法 选取结直肠癌患者200例,收集各研究对象的结直肠癌病理标本和癌旁正常组织各一份,应用免疫组织化学法(S-P法),检测结直肠癌组织和癌旁正常组织中COX-2及Her2/neu蛋白质表达.结果 结直肠癌中Her2/neu表达情况:蛋白质表达阳性占86.0%,明显高于对照组Her/neu阳性表达(24.0%)(P<0.05).结直肠癌中Her2/neu表达与Dukes分期和肝转移相关(P<0.05).结直肠癌中COX-2表达阳性率为89.0%,而癌旁正常组织中其阳性表达率为32.0%,两者差异具有统计学意义(P<0.05),过表达与肿瘤肝转移和Dukes分期有关(P<0.05),与Her2/neu表达有正相关性(P<0.05).结论 采用免疫组织化学法探究蛋白质的表达,结肠癌COX-2及Her2/neu蛋白质的表达率高于癌旁正常组织;COX-2及Her2/neu蛋白质的表达与肝转移和Dukes有关,而且它们之间的表达具有正相关性(P<0.05).     

Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines

Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G(1)/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.     

SCA-1 Identifies the Tumor-Initiating Cells in Mammary Tumors of BALB-neuT Transgenic Mice

Cancer stem cells, initiating and sustaining the tumor process, have been isolated in human and murine breast cancer using different cell markers. In the present study, we aimed to evaluate the presence and characteristics of stem/tumor-initiating cells in the model of the mouse mammary neoplasia driven by the activated form of rat Her-2/neu oncogene (BALB-neuT mice). For this purpose, we generated tumor spheres from primary spontaneous BALB-neuT tumors. Tumor sphere cultures were characterized for clonogenicity, self-renewal, and ability to differentiate in epithelial/myoepithelial cells of the mammary gland expressing basal and luminal cytokeratins and alpha-smooth muscle actin. In addition, tumor spheres were more resistant to doxorubicin compared with parental tumor cells. In the attempt to identify a selected marker for the sphere-generating cells, we found that Sca-1⁺ cells, present in tumors or enriched in mammospheres, and not CD24⁺ or CD29⁺ cells, were responsible for the sphere generation in vitro. Moreover, cells from the tumor spheres showed an increased tumor-generating ability in respect to the epithelial tumor cells. Sca-1⁺ sorted cells or clonal mammospheres derived from a Sca-1⁺ cell showed a superimposable tumor-initiating ability. The data of the present study indicate that a Sca-1⁺ population derived from mammary BALB-neuT tumors is responsible for sphere generation in culture and for initiating tumors in vivo.     

Her2/neu induces all-trans retinoic acid (ATRA) resistance in breast cancer cells

We observed that all-trans retinoic acid (ATRA) inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transactivating ligand HEREGULIN. This suggests that Her2/neu causes breast cancer cells to be resistant to the growth inhibitory effects of ATRA. To confirm this observation, MDA-MB-453 and BT-474 cells, which have high levels of Her2/neu and are resistant to ATRA, were incubated with the trastuzumab (Herceptin) antibody so that we could determine whether inhibition of the expression and function of Her2/neu would resensitize these cells to ATRA. Indeed, we found that MDA-MB-453 and BT-474 cells treated with trastuzumab were growth inhibitory by ATRA. We then determined whether Her2/neu uses Grb2 and Akt proteins to induce ATRA resistance. Liposome-incorporated Grb2 antisense oligonucleotides (L-Grb2) and a dominant negative (DN) AKT mutant were used to down-regulate Grb2 expression and inhibit Akt activity, respectively. When incubated with L-Grb2 or transfected with the DN AKT mutant, ATRA-resistant, Her2/neu-overexpressing cells became sensitive to ATRA. Our results indicate that Her2/neu utilizes Grb2 and Akt proteins to induce ATRA resistance in breast cancer cells. ATRA sensitivity was also correlated with RARalpha protein levels since higher RARalpha protein levels were observed in cells in which the Her2/neu pathway was inhibited.     

Targeting Her-2/neu in breast cancer: as easy as this!

Her-2/neu-positive tumors account for approximately 20% of all breast cancer and these tumors carry poor prognosis. Trastuzumab and lapatinib are 2 agents that have gained FDA approval for treating Her-2/neu-positive breast cancer. Trastuzumab has been shown to improve all endpoints when added to chemotherapy compared to chemotherapy alone, both in the adjuvant and metastatic phases. The addition of lapatinib to capecitabine has recently been shown to improve time to progression in trastuzumab-refractory patients with unique activity against brain metastasis. In spite of their impressive results, a significant fraction of patients still develop either primary or secondary resistance, a fact that entails the discussion of possible mechanisms of resistance. Biomarkers including PTEN, p95HER2, IGF1R and others have been linked to response to Her-2/neu-targeting agents. In this article, we overview the Her-2/neu signaling pathways and how a better understanding of the different molecular aspects of this oncogene could serve in optimizing the use of Her-2/neu-targeting agents. We also discuss the preclinical and clinical data of these biomarkers that may guide clinicians in choosing the right drug whenever possible.     

Targeting her-2/neu with antirat Neu virosomes for cancer therapy.

HER-2/neu (p185(HER2)) oncogene represents an attractive target for antibody-mediated immunotherapy. The major problem of combining chemotherapy and immunotherapy is the severe side effects that limit the use of doxorubicin (Doxo) as a cytotoxic drug. We have used virosomes (Vir; reconstituted fusion-active viral envelopes) as a new drug delivery system and have shown that Vir are capable of binding and penetrating into tumor cells, delivering cytotoxic drugs. We have additionally demonstrated that conjugating Fab' fragments of an antirat Neu (anti-rNeu) monoclonal antibody to Vir selectively and efficiently inhibits tumor progression of established rNeu-overexpressing breast tumors. Fab'-Doxo-Vir combine the antiproliferative properties of the monoclonal antibody and the cytotoxic effect of Doxo in vivo. Furthermore, Fab'-Doxo-Vir significantly inhibit tumor formation at a tumor load representing metastatic spread. These results indicate that Vir conjugated with an antibody against a tumor antigen are a promising new selective drug delivery system for the treatment of tumors expressing a specific tumor antigen.     

Tamoxifen resistance and Her2/neu expression in an aged, irradiated rat breast carcinoma model

Clear links have been established between occupational or therapeutic radiation exposure and breast cancer. Tamoxifen chemoprevention following radiation exposure may be able to reduce the risk of developing breast cancer later in life. In order to model carcinogenesis in this setting, an in vivo model of tamoxifen chemoprevention and tamoxifen failure in a radiation-induced rat mammary carcinoma model was characterized. Two hundred and twenty-seven 60-day-old female rats received whole body or sham exposure to ionizing radiation. Thirty days later long-term, continuous, tamoxifen chemoprevention was initiated in half the population and all animals were monitored over three and a half years for the development of mammary tumors. Mammary tumors were surgically removed and carcinomas were histologically identified and characterized. Results showed that tamoxifen chemoprevention decreased the incidence and prolonged the latency of radiation-induced mammary carcinomas. However, many individuals receiving tamoxifen chemoprevention developed their first carcinoma very late in life. These carcinomas shared morphological features distinct from the majority of carcinomas that developed in the absence of tamoxifen chemoprevention. Analyses of cell lines established from these carcinomas and immunohistochemistry of tumor sections revealed that the highest levels of Her2/neu expression were associated with in vivo tamoxifen exposure. Treatment of rat mammary carcinoma cells with an anti-rat Her2/neu monoclonal antibody (MAb 7.16.4) inhibited cell growth and this effect was more pronounced in the presence of tamoxifen. These studies suggest that carcinoma growth driven by the Her2/neu pathway may be associated with tamoxifen chemoprevention failure in the rat mammary carcinoma model. Additionally, strategies combining targeted Her2/neu antibodies, vaccines or drugs with estrogen pathway modification may be more effective in reducing breast cancer chemoprevention failures.