HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification.

Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells. By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/ HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.     

HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway

Overexpression of HER-2/neu correlates with poor survival of breast and ovarian cancer patients and induces resistance to tumor necrosis factor (TNF), which causes cancer cells to escape from host immune defenses. The mechanism of HER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-kappaB without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes the HER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibits IkappaB kinases, IkappaB phosphorylation, and NF-kappaB activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-kappaB anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia.     

Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface.     

Spontaneous Epithelial-Mesenchymal Transition and Resistance to HER-2-Targeted Therapies in HER-2-Positive Luminal Breast Cancer

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.     

Role of HER-2/neu signaling in sensitivity to tumor necrosis factor-related apoptosis-inducing ligand: enhancement of TRAIL-mediated apoptosis by amiloride

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in numerous transformed cell lines but not in most normal cells. Although this selectivity offers a potential therapeutic application in cancer, not all cancers are sensitive to TRAIL-mediated apoptosis. In this study, we observed that amiloride, a current clinically used diuretic drug, which had little or no cytotoxicity, sensitized TRAIL-resistant human prostate adenocarcinoma LNCaP and human ovarian adenocarcinoma SK-OV-3 cells. The TRAIL-mediated activation of caspase, and PARP cleavage, were promoted in the presence of amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (DR4, DR5, and DcR2) and anti-apoptotic proteins (FLIP, IAP, and Bcl-2). However, amiloride dephosphorylated HER-2/neu tyrosine kinase as well as Akt, an anti-apoptotic protein. Interestingly, amiloride also dephosphorylated PI3K and PDK-1 kinases along with PP1alpha phosphatase. In vitro kinase assay revealed that amiloride inhibited phosphorylation of kinase as well as phosphatase by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the HER-2/neu-PI3K-Akt pathway-associated kinases and phosphatase.     

HER-2: a biomarker at the crossroads of breast cancer immunotherapy and molecular medicine

The oncoprotein encoded by the HER-2 oncogene is a member of the HER family of receptor tyrosine kinases and is actually the first successfully exploited target molecule in new biomolecular therapies of solid tumors. The association of HER-2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapy. In addition, HER-2 overexpression fosters its immunogenicity, as shown by the frequency of B and T cell-mediated responses against this oncoprotein in cancer patients, and it is being investigated as a promising molecule for either passive and active immunotherapy strategies. This review summarizes a number of immune intervention approaches that target HER-2 in breast cancer.     

Breast cancer: the upgraded role of HER-3 and HER-4

Breast cancer is the most common cancer in women and the ErbB receptor family holds crucial role in its pathogenesis. Among them, epidermal growth factor receptor and HER-2 are the most studied members and their overexpression has been associated with aggressive clinical behaviour. These data were further strengthened by the clinical success of trastuzumab, a monoclonal antibody against HER-2 in breast cancer patients with HER-2 overexpression and/or amplification. However, trastuzumab failure in some patients may partly be attributed to co-expression of other ErbB receptors. Herein, we provide updated views regarding the role of HER-3 and HER-4 in breast cancer. Accumulated evidence implies that these receptors should be considered more than heterodimerisation partners. Their expression profile might be useful in predicting responsiveness to current treatment options, while new strategies targeting their ligands and downstream effectors are being developed.     

A critical role for HSP90 in cancer cell invasion involves interaction with the extracellular domain of HER-2

HSP90 is a ubiquitously expressed molecular chaperone that controls the folding, assembly, intracellular disposition, and proteolytic turnover of many proteins, most of which are involved in signal transduction processes. Recently, a surface form of HSP90 has been identified and associated with cell migration events. In this paper, we explore the interaction of surface HSP90 with HER-2, a receptor-like glycoprotein and member of the ErbB family of receptor tyrosine kinases that play central roles in cellular proliferation, differentiation, and migration as well as in cancer progress. The involvement of HSP90 in the regulation of HER-2 has been attributed so far to receptor stabilization via interaction with its cytoplasmic kinase domain. Here we present evidence, using glutathione S-transferase pull-down and transfection assays, for a novel interaction between surface HSP90 and the extracellular domain of HER-2. Specific disruption of this interaction using mAb 4C5, a function-blocking monoclonal antibody against HSP90, inhibits cell invasion accompanied by altered actin dynamics in human breast cancer cells under ligand stimulation conditions with heregulin. Additionally, disruption of surface HSP90/HER-2 interaction leads to inhibition of heregulin-induced HER-2-HER-3 heterodimer formation, reduced HER-2 phosphorylation, and impaired downstream kinase signaling. Interestingly, this disruption does not affect HER-2 internalization. Our data suggest that surface HSP90 is involved in heregulin-induced HER-2 activation and signaling, leading to cytoskeletal rearrangement, essential for cell invasion.     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

Tumor-targeting,systemically delivered antisense HER-2 chemosensitizes human breast cancer xenografts irrespective of HER-2 levels

BACKGROUND: The failure to respond to chemotherapy is a major obstacle in the successful treatment of breast cancer. We have previously shown that anti-HER-2 antisense oligonucleotide (AS HER-2 ODN) treatment was able to sensitize breast cancer cells to various chemotherapeutic agents in vitro irrespective of their HER-2 status, indicating that the use of AS HER-2 ODN therapy for breast cancer is not limited to tumors overexpressing the protein. One of the main drawbacks to the use of antisense therapy in the clinical setting is the lack of an efficient, tumor-targeting, systemic delivery method. We have developed a tumor-specific, ligand-targeting, cationic liposome delivery system designed for systemic gene therapy of cancer. In this study we employ this ligand-liposome strategy to enhance the delivery of the AS Her-2 ODN to breast cancer cells, including those that do not overexpress HER-2, in vitro and in vivo. MATERIALS AND METHODS: A cationic liposome complex that includes folate as the targeting ligand was designed and optimized for more efficient delivery of AS HER-2 ODN to breast tumors cells in vitro, and more significantly, for systemic delivery with tumor-specific targeting in vivo. Human breast cancer cell line MDA-MB-435, which does not overexpress HER-2, was used to compare the degree of chemosensitization to the taxanes of AS HER-2 ODN delivered via the optimized folate-liposome versuscommercial Lipofectin. MDA-MB-435 xenograft tumors were also used to evaluate the anti-tumor effect of the combination of systemically delivered folate-liposome-AS HER-2 ODN and docetaxel (Taxotere). RESULTS: The optimized folate-liposome-AS HER-2 ODN complex significantly increases the response of breast tumor cell lines to conventional chemotherapeutic agents in vitro as compared to AS HER-2 delivered via an unliganded commercially available reagent, Lipofectin. In vivo, the folate-liposome-AS HER-2 ODN complex has prolonged stability in blood and increased uptake in tumors. More significantly, the combination of intravenously administered ligand-liposome-AS HER-2 ODN and docetaxel resulted in a marked inhibition of xenograft growth in an aggressive breast cancer model that does not overexpress HER-2, even after treatment ended. CONCLUSIONS: Although there are other reports of liposomal delivery of AS ODNs, this is the first report of in vivo efficacy against human cancer cells using a tumor-targeting liposome delivery system for systemic AS therapy. Moreover, the increased stability in circulation and anti-tumor efficacy observed were obtained without the need for continuous intravenous infusion. HER-2 is an integral component within a network of cell growth pathways that can affect many different types of tumors where HER-2 may be a contributing factor, such as ovarian, esophageal, and GI malignancies including colon and pancreatic cancers. Therefore, the effectiveness of this therapy with xenograft tumors that do not overexpress HER-2 has the potential to expand the clinical usefulness of this efficacious form of therapy.     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis>(776-90) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/<Emphasis Type="Italic">neu</Emphasis>+ tumors

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay

PURPOSE: The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). EXPERIMENTAL DESIGN: Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). RESULTS: Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). CONCLUSIONS: ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.     

CXCR4的抑制性多肽对乳腺癌细胞CXCR4和HER-2表达及HERCEPTIN药物敏感性的影响

目的 探讨CXCR4抑制性多肽对人乳腺癌细胞株SKBR3膜受体HER-2和CXCR4的蛋白表达及其对Herceptin药物敏感性的影响.方法 抑制性多肽、Herceptin单独或联合处理乳腺癌SKBR3细胞24、48h后,用MTT法观测SKBR3细胞的增殖抑制效应;Weatern blot和免疫纽化法检测SKBR3细胞中CXCR4和HER-2蛋白表达;RT-PCR检测HER-2、CXCR4 mRNA的表达,流式细胞仪检测细胞周期的变化.结果 多肽和Herceptin可不同程度地下调人乳腺癌细胞SKBR3中CXCR4和HER-2的蛋白表达.多肽对细胞生长抑制不明显,与Herceptin联合用药后,生长抑制率高于对照组和Herceptin单独用药组(P<0.001),48h生长抑制率为57.94%±5.3,且呈时问依赖(P<0.05),Herceptin单独作用SKBR3-细胞,细胞周期阻滞于G0/GI期,并且S期的比例减少,与多肽联合作用后,细胞周期又进一步阻滞于G2/M期,S期细胞进一步降低.结论 抑制性多肽能不同程度地下调膜受体HER-2和CXCR4的表达,提高人乳腺癌细胞株SKBR3对Herceptin药物的敏感性.     

HER-2/neu raises SHP-2, stops IFN-gamma anti-proliferation in bladder cancer

Gene amplification or HER-2/neu protein overexpression signals a poor outcome for bladder cancer patients. We investigated the anti-proliferative effect of IFN-gamma in HER-2/neu-transfected human bladder cancer cells (TCC-N5 and TCC-N10). The cells continued growing after IFN-gamma stimulation but did not activate the Janus kinase (Jak)/Stat pathway. We found Jak/Stat protein phosphatase in TCC-N5 and TCC-N10 cells with upregulated Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). After the cells had been treated with AG825, a HER-2/neu-specific inhibitor, SHP-2 expression declined, and Jak2/Stat1 reactivated. Similar results were reported in a mouse bladder cancer cell line, MBT2, with constitutive HER-2/neu overexpression. Further, AG825 pretreatment restored the anti-proliferation activity of IFN-gamma in TCC-N5 and TCC-N10 cells. Therefore, the suppression of IFN-gamma signaling in HER-2/neu-overexpressing bladder cancer cells might be due to SHP-2 upregulation. The regulation of SHP-2 by HER-2/neu provides a new target for blocking the HER-2/neu oncogenic pathway.     

HER-2 diagnostics

HER-2 (c-erbB2, neu) receptor is the molecular marker of ductal breast cancer although it is verexpressed in other adenocarcinoma as well (e.g.endometrial, colorectal and lung cancers). The increased receptor expression is most frequently (90-97%) due to gene amplification. Detection of the overexpression of HER-2 helps to determine prognosis, to predict chemoresistance and to select for Herceptin therapy. HER-2 overexpression can be estimated either by immunohistochemistry or by fluorescent in situ hybridisation (FISH). Standardization of the immunohistochemical HER-2 tests is the best in HercepTest DAKO), however, the frequent 2+level requires complementary FISH test to verify gene amplification. This combination is not necessary at low (0-1+) or high (3+) level of immunohistochemical reactions, because the correlation with gene amplification status is acceptably high. Recently several new anti-HER-2 antibodies have been introduced into HER-2 diagnostics in various countries. According to our experiences we recommend to combine rationally the immunohistochemistry and FISH techniques to determine the HER-2 status in breast cancer.     

Immunogenic HER-2/neu peptides as tumor vaccines

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004