ErbB2-overexpressing Cancer Cells Research Articles

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

HER2/ErbB2 activates HSF1 and thereby controls HSP90 clients including MIF in HER2-overexpressing breast cancer

Overexpression of the human epidermal growth factor receptor-2 (HER2) in breast cancer strongly correlates with aggressive tumors and poor prognosis. Recently, a positive correlation between HER2 and MIF (macrophage migration inhibitory factor, a tumor-promoting protein and heat-shock protein 90 (HSP90) client) protein levels was shown in cancer cells. However, the underlying mechanistic link remained unknown. Here we show that overexpressed HER2 constitutively activates heat-shock factor 1 (HSF1), the master transcriptional regulator of the inducible proteotoxic stress response of heat-shock chaperones, including HSP90, and a crucial factor in initiation and maintenance of the malignant state. Inhibiting HER2 pharmacologically by Lapatinib (a dual HER2/epidermal growth factor receptor inhibitor) or CP724.714 (a specific HER2 inhibitor), or by knockdown via siRNA leads to inhibition of phosphoactivated Ser326 HSF1, and subsequently blocks the activity of the HSP90 chaperone machinery in HER2-overexpressing breast cancer lines. Consequently, HSP90 clients, including MIF, AKT, mutant p53 and HSF1 itself, become destabilized, which in turn inhibits tumor proliferation. Mechanistically, HER2 signals via the phosphoinositide-3-kinase (PI3K)–AKT– mammalian target of rapamycin (mTOR) axis to induce activated pSer326 HSF1. Heat-shock stress experiments confirm this functional link between HER2 and HSF1, as HER2 (and PI3K) inhibition attenuate the HSF1-mediated heat-shock response. Importantly, we confirmed this axis in vivo. In the mouse model of HER2-driven breast cancer, ErbB2 inhibition by Lapatinib strongly suppresses tumor progression, and this is associated with inactivation of the HSF1 pathway. Moreover, ErbB2-overexpressing cancer cells derived from a primary mouse ErbB2 tumor also show HSF1 inactivation and HSP90 client destabilization in response to ErbB2 inhibition. Furthermore, in HER2-positive human breast cancers HER2 levels strongly correlate with pSer326 HSF1 activity. Our results show for the first time that HER2/ErbB2 overexpression controls HSF1 activity, with subsequent stabilization of numerous tumor-promoting HSP90 clients such as MIF, AKT and HSF1 itself, thereby causing a robust promotion in tumor growth in HER2-positive breast cancer.

The Oncogene HER2/neu (ERBB2) Requires the Hypoxia-inducible Factor HIF-1 for Mammary Tumor Growth and Anoikis Resistance

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

ErbB2 Dephosphorylation and Anti-Proliferative Effects of Neuregulin-1 in ErbB2-Overexpressing Cells; Re-evaluation of Their Low-Affinity Interaction

Neuregulin-1 binds to ErbB3 and ErbB4 and regulates cancer proliferation and differentiation. Neuregulin-1 had been suggested to also react with ErbB2, but this argument becomes controversial. Here, we re-evaluated the cellular responses and ErbB2 interaction of neuregulin-1 in ErbB2 overexpressing cell lines. In a competitive ligand-binding assay, we detected significant replacement of [35S]-labeled neuregulin-1 with nano molar ranges of cold neuregulin-1 in L929 cells expressing ErbB2 alone and SKOV3 cells carrying sulf-1 cDNA but not in these parental cells. The concentration of neuregulin-1 significantly decreased thymidine incorporation and phosphorylation of ErbB2 (Tyr877, Tyr1396, and Tyr1121) in ErbB2-overexpressing cancer cells as well as in L929 cells expressing ErbB2. A crosslinking assay ascertained the presence of neuregulin-1 immunoreactivity in the ErbB2 immune complexes of L929 expressing ErbB2 alone. These results suggest that the higher concentrations of neuregulin-1 exert an anti-oncogenic activity to attenuate ErbB2 auto-phosphorylation potentially through its low-affinity interaction with ErbB2.

Abstract 3208: Overcoming cancer therapeutic resistance by targeting dysregulated glucose metabolism

Abstract Heat shock transcription factor 1 (HSF1) is the master regulator of the heat shock responses in eukaryotes. Upon exposure to a variety of stresses, HSF1 activates its target genes, subsequently activating the heat shock responses. Although not well understood, the functions of HSF1 are far beyond the classical induction of heat shock responses. Recent reports have shown that HSF1 plays an important role in cancer development. We investigated the role and mechanism of HSF1 in regulating oncogene-mediated dysregulated cellular metabolism and cell growth in cancer cells. We found that oncogene ErbB2-overexpressing cancer cells possessed significantly higher level of glycolysis when compared to ErbB2-low expressing cells, and the downregulation of ErbB2 markedly decreased glycolysis. Overexpression of ErbB2 increased the expression of HSF1 and two critical glycolysis-regulating molecules, lactate dehydrogenase A (LDH-A) and Hexokinase (HK). ErbB2 activated HSF1, indicated by the increased HSF1 trimer formation and DNA binding activity, and promoted the rate of HSF1 protein translation. HSF1 bound to the promoter of LDH-A and transcriptionally activated LDH-A. Meanwhile, the downregulation of HSF1 reduced the expression of LDH-A and subsequently decreased cancer cell glycolysis and growth. These results demonstrate that in cancer cells, ErbB2 promotes glycolysis and cell growth through HSF1-mediated upregulation of LDH-A. Trastuzumab (Herceptin) and Paclitaxel (Taxol) are important drugs in treatment of breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. We investigated the antitumor effects of trastuzumab and Taxol in combination with glycolysis inhibitors in breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of HSF1 and LDH-A in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab and Taxol resistance. Strikingly, we found that combining trastuzumab or Taxol with glycolysis inhibition synergistically inhibited both drug-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. These results show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance. This work was supported by NIH Grant RO1CA149646, The Vincent F. Kilborn, Jr. Cancer Research Foundation, and Radiumhospitalets Legater Project 334003. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3208. doi:1538-7445.AM2012-3208

Synergistic Enhancement of the Potency and Selectivity of Small Molecule Transcriptional Inhibitors

In spite of their considerable therapeutic potential, the development of highly potent and selective transcriptional inhibitors has proven elusive. We demonstrate that combinations of transcriptional inhibitors of erbB2 expression and existing therapeutic agents that target erbB2 activity and lifetime lead to a synergistic increase in activity, with dose reductions as high 30 fold compared to individual agents. The synergy is selective for erbB2 overexpressing cancer cells. These results highlight the potential of a generalizable approach that will improve the utility of transcriptional inhibitors as both biochemical tools and potential therapeutics.

Abstract 1710: Expression of erbB3 upregulates Survivin via transcription-independent mechanism in erbB2-overexpressing breast cancer cells to confer Paclitaxel resistance

Abstract The erbB2 receptor is often activated via dimerization with another erbB receptor, such as EGFR or erbB3. Studies show that co-expression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB2 requires erbB3 to stimulate breast cancer cell proliferation. Increased resistance to therapeutic agents such as paclitaxel is one of the mechanisms by which erbB2 contributes to breast tumorigenesis. While several molecular mechanisms contributing to paclitaxel resistance have been proposed, a series studies demonstrate that overexpression of erbB2 inhibits paclitaxel-induced apoptosis in breast cancer cells. However, the role of erbB3 in paclitaxel resistance remains unclear. Our current studies focus on determining the role(s) of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells. Overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific shRNA in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, PARP cleavage, and activation of caspase-3, and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the protein but not mRNA expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific PI-3K, Akt, and mTOR inhibitors, but not the MEK inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition. Our studies demonstrate that heterodimerization of erbB2/erbB3 results in paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway. Elevated expression of erbB3 upregulates Survivin through transcription-independent mechanism in erbB2-overexpressing cancer cells, suggesting that novel strategies targeting erbB3 or Survivin protein translation may enhance the efficacy of chemotherapeutic agents against breast cancer with erbB2-overexpressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2011-1710

Abstract 654: MM-111, an ErbB2/ErbB3 bispecific antibody, effectively combines with lapatinib to inhibit growth of ErbB2-overexpressing tumor cells

Abstract The oncogenic receptor ErbB2 (HER2) is frequently overexpressed in a variety of cancer indications, including breast, gastric, bladder, and lung. ErbB3 (HER3), the preferred dimerization partner of ErbB2, is critical for the survival and growth of ErbB2-overexpressing tumors, but is poorly inhibited by current ErbB2-targeted therapies. MM-111 is a novel bispecific antibody designed to specifically inhibit function of the ErbB2/ErbB3 heterodimer in ErbB2-overexpressing tumors. MM-111 binds simultaneously to ErbB2 and ErbB3 and blocks the interaction of ErbB3 with its ligand, heregulin. Inhibiting the ErbB2/ErbB3 heterodimer prevents ligand-induced activation of the phosphotidylinositol 3-kinase (PI3K) pro-survival pathway, altering cell cycle progression and inhibiting tumor growth. Upregulation of both heregulin and ErbB3 has been observed in tumors that have acquired resistance to the ErbB2-targeted therapies trastuzumab and lapatinib. In addition, ErbB2 and ErbB3 are frequently upregulated in estrogen receptor positive breast cancers that have developed resistance to anti-estrogen therapies. These data indicate that the ErbB2/ErbB3 receptor heterodimer potently activates growth of tumors characterized by ErbB2 overexpression and implicate ErbB3 as a mechanism of resistance to current therapies. Because of its ability to inhibit ErbB3 activity, MM-111 could be effective in combination with ErbB2-targeted therapies by overcoming ErbB3-mediated resistance. Previous data demonstrating that MM-111 acts in concert with trastuzumab to inhibit growth of ErbB2-overexpressing human tumor models support this hypothesis. We now show that MM-111 effectively combines with lapatinib to inhibit growth of tumors driven by the ErbB2/ErbB3 heterodimer. MM-111, as a single agent, is more effective than lapatinib at inhibiting heregulin-driven activation of ErbB3 and AKT in ErbB2-overexpressing cancer cell lines. In these same cell lines, the combination of MM-111 and lapatinib results in greater AKT inhibition than either treatment alone. In addition, we have demonstrated in ErbB2-overexpressing 3D spheroid cancer models that treatment with the MM-111/ lapatinib combination increases the number of cells in early phase apoptosis as compared to single-drug treatments. Finally, in an ErbB2-overexpressing cancer xenograft model, the combination of MM-111 and lapatinib results in greater inhibition of tumor growth than either therapy alone. In conclusion, we show that MM-111 and lapatinib positively combine to inhibit the growth of ErbB2-overexpressing tumors. Our results suggest that concurrent treatment with MM-111 and lapatinib may provide a potent therapeutic regimen for cancer patients whose tumors overexpress ErbB2 by deterring ErbB3-mediated resistance to lapatinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2011-654

Abstract 3485: MM-111, an ErbB2/ErbB3 bispecific antibody with potent activity in ErbB2-overexpressing cells, positively combines with trastuzumab to inhibit growth of breast cancer cells driven by the ErbB2/ErbB3 oncogenic unit

Abstract MM-111 is a novel bispecific antibody fusion protein which targets the ErbB2/ErbB3 oncogenic unit, blocking activation of the phosphatidylinositol 3-kinase (PI3K) pro-survival pathway. The anti-ErbB2 arm of MM-111 binds with high affinity to the ErbB2 receptor, which localizes the bispecific molecule to ErbB2 over-expressing tumor cells and promotes binding of the anti-ErbB3 arm to the ErbB3 receptor. MM-111 binding to ErbB3 results in inhibition of ErbB3 signaling by blocking the binding of the ErbB3 physiological ligand heregulin. MM-111 treatment of ErbB2 overexpressing cancer cells inhibits activation of the PI3K pathway with sub-nanomolar potency, blocks cell cycle progression and attenuates tumor cell growth in multiple xenograft models. ErbB2 over-expressing tumor cells are addicted to growth signals provided by the ligand-activated ErbB2/ErbB3 heterodimer. Activation of downstream PI3K pathway signaling also occurs through ligand-independent ErbB2/ErbB2 homodimers and ErbB2/ErbB3 heterodimers. Recently the ErbB2-targeted therapeutic antibody trastuzumab was shown to inhibit basal ErbB3 signaling in the absence of ligand stimulation, purportedly by interrupting ligand-independent ErbB2/ErbB3 heterodimers formed through overexpression of ErbB2. However, in these studies trastuzumab did not effectively block ligand-induced activation of the ErbB2/ErbB3 oncogenic unit. Indeed, there is emerging evidence that ligand-induced ErbB3 activation may have an important role in resistance to trastuzumab. As MM-111 and trastuzumab have distinct and potentially complimentary effects on signaling in cells overexpressing ErbB2, we hypothesized that their combination may synergistically effect inhibition of tumor cell growth driven by the ErbB2/3 signaling network. Our data demonstrate that MM-111 and trastuzumab positively combine to inhibit breast cancer growth in multiple in vitro breast cancer models expressing ErbB2 and ErbB3. Further, in the BT-474 breast cancer xenograft model the combination of MM-111 and trastuzumab results in greater inhibition of tumor growth and an increased number of completely regressed tumors compared to the monotherapy treatment groups. Pharmacodynamic analysis of samples from these studies show that repeated administration of the combined therapeutic agents results in strong inhibition of the ErbB3/PI3K pathway. In conclusion, we show that MM-111 and trastuzumab inhibit the growth of ErbB2 over-expressing breast tumors with distinct mechanisms that act synergistically in combination. Concurrent treatment with MM-111 and trastuzumab may provide a potent therapeutic regimen for ErbB2-overexpressing breast cancer patients and potentially deter acquired resistance to trastuzumab through ErbB3 activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3485.

Interactions between anti-ErbB2 antibody A21 and the ErbB2 extracellular domain provide a basis for improving A21 affinity

Anti-ErbB2 antibodies are well researched for the therapy of ErbB2-overexpressing tumors. The therapeutic potential and efficacy of these antibodies are closely related to their affinities to ErbB2. Previously we reported that an anti-ErbB2 antibody A21 targeting a conformational epitope comprising several loops in ErbB2 extracellular subdomain I and II could inhibit the proliferation of ErbB2-overexpressing cancer cells in vitro and in vivo. Here we found that another structureless and non-conserved loop in subdomain I of ErbB2 extracellular domain (ECD) was important for binding to A21, and then the antigen-contact sites on A21 were determined by site-directed mutation. The loop was constructed by molecular modeling, and a new model of A21-ErbB2 complex was generated by docking using the crystal structure of the scfv A21 and the model of ErbB2 ECD with the loop built. Based on the complex model, computational design for A21 affinity improvement was performed to enhance its affinity to ErbB2. Two mutants with about 1.7-fold improvement in affinity were obtained. Our study provided a rational molecular basis for affinity improvement and mechanism investigation of A21.

Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody-antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1-192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 A resolution from a single flash-cooled crystal; the crystal belonged to space group P2(1)2(1)2(1).

Spotlight

Bertino et al., pp. 12–20 Like asbestos, the mineral erionite can transform human mesothelial cells, leading to malignant mesothelioma (MM). Unlike asbestos, erionite can do its damage without the aid of SV40, according to this study by Bertino et al. The findings could explain the high rate of MM in the Cappadocia region of Turkey, where SV40 has not been isolated. They also suggest that the hazard of exposure to similar fibers should be carefully evaluated. Erionite, a ubiquitous zeolite, is a strong mutagen and induces a high incidence of MM in rats who inhale it. Exposure to erionite fibers leads to the creation of reactive oxygen metabolites from macrophages and turns up transcription of early response proto-oncogenes. Earlier work has shown that asbestos fibers-induced transformation of cells was enhanced by SV40 infection acting through the PI3K/Akt signaling pathway. In the current study, the authors looked at how efficiently erionite transformed human mesothelial cells in the absence of SV40, compared with two varieties of asbestos – amosite and chrysotile. They also investigated signal transduction in the transformed cells to better understand the mechanism by which the mineral fibers spur transformation. Cells exposed to erionite began proliferating faster within 24 hours. The erionite fibers were less toxic to cells than the amosite, allowing a high proliferation rate. Chrysotile asbestos, although not as toxic to cells as amosite, could not promote proliferation. The cells showed the morphologic changes associated with full transformation. Obviously, erionite transforms cells in the absence of SV40. Gorlova et al., pp. 111–118 Relatives of people who have had lung cancer but have never smoked appear to have a higher risk for cancer, particularly young-onset cancers, according to a new case-control study from Gorlova et al. The study, which included nearly 5,000 people, contradicts some earlier reports and could indicate that shared genetic factors could predispose individuals to lung cancer and several other cancers. Several previous studies have reported no increased risk of lung cancer in immediate relatives of lung cancer patients who have not smoked. One study, based on a small number of participants, reported a 6-fold increase in lung cancer patients. Because individuals who have never smoked and who develop lung cancer may represent a distinct, genetically susceptible population, identifying familial aggregation of cancers could help illuminate genetic factors that contribute to lung cancer susceptibility. Gorlova et al. analyzed data from 2,465 relatives of 316 nonsmoking lung cancer patients and 2,441 relatives or 318 never-smoking controls to compare the groups' risk of lung cancer and other cancers. The authors identified a 25% increase in risk of any type of cancer among first-degree relatives of lung cancer patients and a 44% increase in risk of cancer occurring before age 50, compared with relatives of controls. Certainly, shared environmental factors, such as secondhand smoke from parental smoking, could cause multiple family members to develop cancer with no shared genetic vulnerability. In this study, there was a slightly higher number of smokers among relatives of controls than lung cancer cases, and parental smoking does not seem to influence the conclusions. The authors controlled for smoking status and conducted analyses stratified by smoking status, and therefore conclude that genetic factors remain the more likely explanation for the familial aggregation of cancers that they observed. Wang et al., pp. 157–164 The one-two punch of rapamycin and herceptin administered together fights breast cancer better than rapamycin alone, says a new study from Wang et al. The results, which confirm earlier work showing increased susceptibility of certain breast cancer cells to inhibitors of the PI3 kinase signaling pathway – such as rapamycin – could improve breast cancer treatment strategies. About a third of breast cancers overexpress ErbB2, a member of the epidermal growth factor receptor (EGFR) family. Overexpression of ErbB2 is associated with a high relapse rate and poor clinical prognosis, and ErbB2 plays a functional role in cell transformation and mammary cell carcinogenesis. Breast cancer cells that overexpress ErbB2 are particularly sensitive to rapamycin, which interferes with cell cycle progression and halts tumor growth. Rapamycin and its analogs are being developed as anticancer drugs alone and in combination with other agents. In this study, the authors investigated rapamycin's effectiveness when paired with herceptin, a monoclonal antibody against ErbB2. In clinical trials, herceptin achieves a 15-30% response rate in breast cancers overexpressing ErbB2. The new results demonstrate that rapamycin and herceptin together inhibit the growth of ErbB2-overexpressing cancer cells in culture and in mouse xenograft and transgenic models more effectively than each component by itself.

A Peptide Conjugate of Vitamin E Succinate Targets Breast Cancer Cells with High ErbB2 Expression

Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic alpha-tocopheryl succinate (alpha-TOS). Treating erbB2-low or erbB2-high cells with alpha-TOS induced similar levels of apoptosis, whereas alpha-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. alpha-TOS rapidly accumulated in erbB2-high cells exposed to alpha-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by alpha-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. alpha-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting alpha-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors.

Peptide Aptamers with Binding Specificity for the Intracellular Domain of the ErbB2 Receptor Interfere with AKT Signaling and Sensitize Breast Cancer Cells to Taxol

The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol.

The level of erbB2 expression predicts sensitivity to the cytotoxic effects of an intracellular anti-erbB2 sFv.

We have previously demonstrated that an intracellular antibody (sFv) directed against erbB2 can achieve a specific cytotoxicity in erbB2 overexpressing cancer cells of varying histogenesis. In order to further delineate the mechanistic basis of the induced apoptosis, transient and stable cotransfections were performed. Transient cotransfection of erbB2 mutant and chimeric molecules demonstrated that the cytoplasmic domain of erbB2, or the homologous cytoplasmic domain of the epidermal growth factor receptor, is required for apoptosis induction. These results were confirmed in assays utilizing differential derivation of stable clones. To examine the effects of varying ratios of the anti-erbB2 sFv and its target erbB2 we performed additional cotransfection experiments in erbB2 negative target cells. When erbB2 levels are held constant, observed cytotoxicity is proportional to the amount of sFv added. In addition, when sFv levels are held constant, increasing levels of cotransfected erbB2 can overcome the apoptotic response. These results indicate that a minimal threshold level of the sFv and its target are required to induce cytotoxicity. To examine this phenomenon in an erbB2 positive cell line, SKOV3 ovarian carcinoma cells were utilized to derive a stable clone expressing low levels of sFv. When this cell line was compared to the parental SKOV3 cell line, it was shown that less exogenous sFv was needed to induce cytotoxicity in the clone already expressing low levels of sFv, indicating that endogenous and exogenous levels of sFv are additive. In summary, the results presented here indicate that the carboxy-terminus of the intracellular domain of the erbB2 molecule is involved in the induction of apoptosis. Furthermore, the expression levels of the sFv and its target protein need to overcome a threshold level in order to achieve a cytotoxic response.

Multiple Grb2-protein complexes in human cancer cells.

Grb2 is an SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the ras signaling pathway. The Grb2-SH2 domain binds phosphotyrosine sequences on activated tyrosine kinases, and one target of the SH3 domains is the ras-nucleotide-exchange factor Sos1. We have examined Grb2-protein interactions in human cancer cells that over-express the receptor tyrosine kinase erbB2. Our results show that the 2 Grb2-SH3 domains complex with Sos1, dynamin and at least 4 other proteins (p228, p140, p55, p28) in these cells. The 2 Grb2-SH3 domains bind these proteins differently, with the N-terminal SH3 domain interacting preferentially with p228, Sos1, p140 and dynamin. The C-terminal SH3 domain has higher affinity toward p28. The Grb2-SH3 domain interactions appear to be similar in erbB2 over-expressing breast, ovarian and lung cancer cells. Also, the major tyrosine-phosphorylated proteins that associate with Grb2 in erbB2 over-expressing cancer cells appear to be erbB2 and Shc. The multiple Grb2-SH3 domain interactions in these cells may mediate novel cellular functions.

Intracellular single-chain antibody directed against ERBB-2 down-regulates cell surface ERBB-2 and selectively eradicates ERBB-2 over-expressing cancer cell lines

Intracellular single-chain antibody directed against ERBB-2 down-regulates cell surface ERBB-2 and selectively eradicates ERBB-2 over-expressing cancer cell lines