HER2 Receptor Tyrosine Kinase Research Articles

Phosphorylation of signal transducer and activator of transcription 3 (STAT3) correlates with Her-2 status, carbonic anhydrase 9 expression and prognosis in esophageal cancer

The signal-transcriptional factor signal transducer and activator of transcription (STAT3) is overexpressed in various tumor entities and promotes tumor progression and metastasis. The tyrosine-kinase receptor Her-2 was shown to activate STAT3-expression and is overexpressed in a subtype of esophageal cancer (EC). STAT3 also regulates carbonic anhydrase IX (CAIX) expression in vivo, promoting IL-6-dependent tumor invasion.Tumor-tissue from 324 patients with EC and 45 patients with precursor lesions were analyzed for the expressions of tyrosine-705 phosphorylated STAT3 (pSTAT3). Data on Her-2-status and CAIX expression were available from previous studies. pSTAT3 was overexpressed in 40 % of adenocarcinomas (AC) and 50 % of squamous cell carcinomas of the esophagus and was associated with worse overall (OS) (p = 0.001) and disease free survival (DFS) (p = 0.001). In Barrett's mucosa without/low-grade dysplasia, pSTAT3 was expressed in 14 % compared to 27 % in patients with high-grade dysplasia (p = 0.018). A significant association between Her-2 and pSTAT3 was found in ACs (p = 0.013), showing that patients with tumors expressing both proteins have significantly worse OS (p = 0.0039) and DFS (p = 0.029). One hundred-fifty (46.3 %) cancer cases were considered as positive for CAIX expression, and a strong correlation between pSTAT3 and CAIX expression was seen (p < 0.001). pSTAT3 plays an important role in the development of AC. Expression of pSTAT3 correlates with Her-2 status and CAIX expression and is associated with tumor progression and worse outcome, offering expectantly therapeutical implications.

Expression of Cysteine Protease Cathepsin L is Increased in Endometrial Cancer and Correlates With Expression of Growth Regulatory Genes

Proteases contribute to tumor invasion and metastasis by degrading basement membranes and extracellular matrix (ECM). In this study, we compared gene expression levels of two proteases, cysteine protease Cathepsin L2 (CTSL2) and matrix metalloproteinase MMP11, in human endometrium and endometrial cancer. Our data demonstrate CTSL2 transcript levels to be strongly elevated in endometrial cancer, particularly in G3 tumors. Furthermore, we observed a highly significant positive correlation of CTSL2 with expression of growth regulatory genes Ki-67, cyclin B1, MYBL2, p21/WAF, and HER2 receptor tyrosine kinase. Our data suggest that CTSL2 might be involved in progression of endometrial cancer.

Maximizing Human Epidermal Growth Factor Receptor 2 Inhibition: A New Oncologic Paradigm in the Era of Targeted Therapy

ERBB2 (HER2) amplification in breast cancer is a classic example of oncogene addiction in solid tumor oncology; most human epidermal growth factor receptor 2 (HER2) –positive tumors exhibit dependency on signaling pathways downstream of the HER2 receptor tyrosine kinase, even after developing resistance to anti-HER2 drugs. Trastuzumab is a monoclonal antibody that inhibits HER2 and demonstrates efficacy in both treatment-naive patients and those who progress after a prior trastuzumab-based regimen. Serial administration of anti-HER2 therapies in clinical practice has dramatically altered the natural course of HER2positive breast cancer. Previously considered to have a worse prognosis, women with HER2-amplified breast cancer who receive trastuzumab now experience survival outcomes similar to those with HER2-negative disease. Furthermore, recent studies suggest that dual HER2 inhibition (the administration of two anti-HER2 therapeutics simultaneously) may induce more durable tumor responses than sequential HER2-specific monotherapy. In Journal of Clinical Oncology, Cortes et al demonstrate the value of persistent HER2 blockade by reporting a clinical benefit rate of 41.2% after combined treatment with the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab in patients with evidence of disease progression while receiving either of the two agents alone. This noteworthy observation suggests that the traditional oncologic paradigm—in which most drugs are used only once and discontinued permanently at the first sign of progression—may require modification in the era of targeted therapy. HER2 is a member of the HER family of transmembrane receptors, which also includes epidermal growth factor receptor, HER3 (ERBB3), and HER4 (ERBB4). In normal tissue, HER proteins mediate cell growth signals transmitted between stromal and epithelial cells. Unlike other members of the family, HER2 does not interact directly with the growth factor ligand. Instead, the receptor functions by a ligand-independent dimerization mechanism that primes HER2 for associations with both itself and other HER kinase family members. In HER2-amplified breast cancers, the overabundance of membrane HER2 further potentiates this ligand-independent mechanism, resulting in “addiction” to HER2 signaling for tumor cell survival. In particular, overexpression of HER2 results in increased HER2 homoand heterodimer formation and augmented signaling through intracellular cascades such as the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathways (Fig 1). Contrary to HER2, HER3 has an impaired intracellular kinase and lacks significant biologic activity when expressed alone. However, when HER3 undergoes heterodimerization with HER2, a potent oncogenic signal may result. The HER2:HER3 heterodimer functions as the most transforming and mitogenic receptor complex of the HER family of proteins and is a robust activator of PI3K pathway signaling. The expression of HER3 can be a rate-limiting step for HER2induced cell growth, because HER2-amplified breast tumors undergo apoptotic cell death when HER3 expression is suppressed. Clinically, patients with high levels of HER2:HER3 heterodimers and low levels of HER2 homodimers exhibit a diminished response to trastuzumab therapy. The dependence on HER2 and pathogenesis of HER2: HER3 heterodimers in HER2-amplified breast cancer provide a scientific rationale for targeting both HER2 and HER3 in the clinical setting (Fig 1). Sequential HER2 blockade aims to achieve an extended and diversified therapeutic blockade of this oncogenic receptor tyrosine kinase. However, as seen in the modest activity of singleagent treatment, demonstrated by Cortes et al and others, repetitive HER2-specific monotherapy is often insufficient to engender long-lasting responses in patients who progress while receiving trastuzumab-based regimens. Trastuzumab-DM1 and pan-HER agents are notable exceptions with reasonable singleagent activity, but these drugs are probably more synonymous to combination treatment than to monotherapy. Trastuzumab-DM1 harnesses the effects of HER2 blockade through its trastuzumab component and simultaneously delivers the cytotoxic agent DM1, and pan-HER drugs are known to affect multiple kinases. Several mechanisms of resistance to single-agent HER2 inhibition have been proposed, including alterations affecting the HER2 receptor, increased interactions with both HER proteins and other membrane-bound receptors, and alternative activation of oncogenic signaling networks such as the PI3K pathway. Dual anti-HER2 regimens, in contrast to monotherapy, show excellent synergy and improved outcome in patients previously JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 14 MAY 1

MET Activation Mediates Resistance to Lapatinib Inhibition of HER2-Amplified Gastric Cancer Cells

HER2 amplification is found in more than 15% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2(+) cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte growth factor (HGF) activation of MET receptors rescued cells from lapatinib-induced growth inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET-mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation.

Pharmacological Strategies to Overcome HER2 Cross-Talk and Trastuzumab Resistance

Approximately 20-30% of breast cancers show increased expression of the HER2 receptor tyrosine kinase. Trastuzumab (Herceptin) is a clinically approved anti-HER2 monoclonal antibody. Many patients with HER2-overexpressing metastatic breast cancer respond to trastuzumab; however, a subset display primary drug resistance. In addition, many patients who initially respond to trastuzumab ultimately develop disease progression. Multiple molecular mechanisms contributing to trastuzumab resistance have been proposed in the literature. These mechanisms include cross-signaling from related HER/erbB receptors and compensatory signaling from receptors outside of the HER/erbB family, including receptors for insulin-like growth factor-I, vascular endothelial growth factor, and transforming growth factor beta. The major downstream signaling pathway activated by HER2 cross-talk is PI3K/mTOR, and a potential integrator of receptor cross-talk is Src-focal adhesion kinase (FAK) signaling. PI3K, Src, and FAK have independently been implicated in trastuzumab resistance. In this review, we will discuss pharmacological inhibition of HER2 cross-talk as a strategy to treat trastuzumab-refractory HER2-overexpresssing breast cancer.

HER2/HER3 Signaling Regulates NK Cell-Mediated Cytotoxicity via MHC Class I Chain-Related Molecule A and B Expression in Human Breast Cancer Cell Lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.

OPCML, a novel systems regulator of tyrosine kinase signaling in ovarian and other cancers

OPCML, a GPI anchored tumor suppressor gene is inactivated by somatic methylation in multiple cancers. We previously identified this gene by LOH mapping and demonstrated that it was inactivated by somatic methylation in 80% of ovarian cancers. Restoring OPCML expression by stable transfection suppressed in-vitro growth and in-vivo tumorigenicity. We hypothesized that OPCML might abrogate growth signaling pathways. In SKOV-3 and PEO1, ovarian cancer cell lines with no expression of OPCML, we demonstrated that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1, FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA and shRNA knockdown in normal ovarian surface epithelial cells up-regulates these same RTKs, with no effect on RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 and VEGFR3. shRNA knockdown shows that loss of OPCML accelerates the growth of normal ovarian surface epithelial cells in vitro. Example immunoprecipitation experiments revealed that OPCML binds to EphA2, FGFR1 and HER2 extracellular domains with no such interaction to EGFR, thus OPCML binds directly to RTKS that it negatively regulates. Cotransfection of ECD less and ECD containing Her2/Neu shows that the functional tumor suppressor phenotype of OPCML is mediated via interaction with the ECD. We demonstrate that OPCML is located exclusively in the raft membrane fraction and sequesters RTKs that it binds to the raft fraction, leading to polyubiquitination and proteosomal degradation via a cav-1 endosomal mechanism resulting in systems depletion of this specific RTK repertoire, that does not occur with RTKs that OPCML does not bind. Pulse-chase experiments confirm rapid loss of HER2 in OPCML expressing cells and not in OPCML deficient cells. We demonstrate that OPCML abrogates EGF mediated phosphorylation of FGFR1, HER2 and EGFR and the downstream phosphosignaling of pErk and pAKT. A recombinant modified OPCML-like protein without a GPI anchor, signal peptide or glycosylation was constructed and expressed in E. coli. This rOPCML tumor suppressor protein therapeutic caused growth inhibition by apoptosis in 6/7 ovarian cancer cell lines tested with no effect on OPCML expressing normal ovarian surface epithelium by an identical mechanism to the transfected normal protein. pERk and pAKT inhibition was seen with functional growth inhibition and increased apoptosis in rOPCML treated cells. rOPCML was then injected intraperitoneally twice weekly in two murine intraperitoneal models of ovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profound inhibition of tumour weight, ascites volume and peritoneal dissemination compared with BSA control. Western analysis from rescued tumors confirmed that the same mechanism of specific RTK downregulation was also evident in vivo. In Summary, the OPCML tumour suppressor mediates its suppressor function by systems level negative regulation of at least 5 RTKs and a recombinant modified derivative is a potent tumor suppressor protein therapeutic in-vitro and in-vivo that recapitulates the in-vitro mechanism.

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide–affibody bioconjugate

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu ( TM) and a targeting ligand, an HER2 antibody mimetic-affibody ( AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA: TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAF in vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant ( P < 0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.

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.

Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer.

Although antiestrogen therapies targeting estrogen receptor (ER) α signaling prevent disease recurrence in the majority of patients with hormone-dependent breast cancer, a significant fraction of patients exhibit de novo or acquired resistance. Currently, the only accepted mechanism linked with endocrine resistance is amplification or overexpression of the ERBB2 (human epidermal growth factor receptor 2 [HER2]) proto-oncogene. Experimental and clinical evidence suggests that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway, the most frequently mutated pathway in breast cancer, promotes antiestrogen resistance. PI3K is a major signaling hub downstream of HER2 and other receptor tyrosine kinases. PI3K activates several molecules involved in cell-cycle progression and survival, and in ER-positive breast cancer cells, it promotes estrogen-dependent and -independent ER transcriptional activity. Preclinical tumor models of antiestrogen-resistant breast cancer often remain sensitive to estrogens and PI3K inhibition, suggesting that simultaneous targeting of the PI3K and ER pathways may be most effective. Herein, we review alterations in the PI3K pathway associated with resistance to endocrine therapy, the state of clinical development of PI3K inhibitors, and strategies for the clinical investigation of such drugs in hormone receptor-positive breast cancer.

Dual EGFR/HER2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Withdrawal by Suppressing ErbB3

Patients with recurrent prostate cancer are commonly treated with androgen withdrawal therapy (AWT); however, almost all patients eventually progress to castration resistant prostate cancer (CRPC), indicating failure of AWT to eliminate androgen-sensitive prostate cancer. The overall goal of these studies is to determine whether dual inhibition of the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 would prolong the effectiveness of this treatment in prostate cancer. We used androgen-dependent LNCaP cells and its CRPC sublines LNCaP-AI and C4-2. Additional data were collected in pRNS-1-1 cells stably expressing a mutant androgen receptor (AR-T877A), and in nude mice harboring CWR22 tumors. Studies utilized EGFR inhibitors erlotinib and AG1478, and HER2 inhibitors trastuzumab and AG879. Dual EGFR/HER2 inhibition induced apoptosis selectively in androgen-sensitive prostate cancer cells undergoing AWT, but not in the presence of androgens, or in CRPC cells. We show that AWT alone failed to induce significant apoptosis in androgen-dependent cells, due to AWT-induced increase in HER2 and ErbB3, which promoted survival by increasing Akt phosphorylation. AWT-induced ErbB3 stabilized the AR and stimulated PSA, while it was inactivated only by inhibition of both its dimerization partners EGFR and HER2 (prostate cancer cells do not express ErbB4); but not the inhibition of any one receptor alone, explaining the success of dual EGFR/HER2 inhibition in sensitizing androgen-dependent cells to AWT. The effectiveness of the inhibitors in suppressing growth correlated with its ability to prevent Akt phosphorylation. These studies indicate that dual EGFR/HER2 inhibition, administered together with AWT, sensitize prostate cancer cells to apoptosis during AWT.

Highlights of This Issue

Administration of recombinant human erythropoietin (rHuEPO) has largely replaced bloodtransfusion in patients with cancer-induced anemia. Potential detrimental effects of thisapproach, such as thromboembolic risks and potential tumor response leading to enhanced disease progression, have become points of discussion and concern. Hedley and colleagues present data demonstrating potentiating effects on metastasis and reduction of chemotherapeutic efficacy in secondary sites by rHuEPO. This study begins to uncover the underlying functional explanation for the adverse events and decreased survival that have been observed in erythropoietin-treated metastatic breast cancer patients undergoing chemotherapy.Cancer-initiating cells (CIC) are being readily identified by elevated levels of aldehyde dehydrogenase (ALDH), namely ALDH1A1, a tumorassociated antigen recognized by HLA class I—restricted CD8+ T cells. Pertinent to developing immunotherapy for targeting CICs, Visus and colleagues demonstrate that ALDHbright cells are sensitive to cytolysis by ALDH1A1-specific CTLs in vitro and in vivo. In preclinical models of human tumor xenografts growing in immunodeficient mice, adoptive therapy with ALDH1A1-specific CD8+ T cells targeted ALDHbright cells and inhibited xenograft growth and metastasis or prolonged survival. These results highlight ALDH1A1 as a target of T cell—based immunotherapy to eliminate CICs.Androgen withdrawal therapy (AWT) is considered the standard of care for patients withmetastatic prostate cancer, but resistance to this therapy develops within 2 to 3 years after treatment. Chen and colleagues show that during AWT, increased levels of the receptor tyrosine kinases HER2 and ErbB3, members of the epidermal growth factor receptor (EGFR)family, result in a significant ErbB-dependent survival advantage that allows progressionto castrationresistant prostate cancer. However, dual EGFR/HER2 inhibition, which alsoinhibits their dimerization partner, ErbB3, induced apoptosis in cells undergoing AWT. Theseresults indicate that administration of dual EGFR/HER2 inhibitors together with AWT may impede the onset of castration-resistant prostate cancer.MicroRNAs (miRNAs) have critical functions across various biologic processes and can be potentially used as cancer biomarkers. Colorectal cancer is a heterogeneous disease, and the identification of various types of colorectal cancer with histopathologic methods alone is challenging. In this study, Balaguer and colleagues used miRNA expression profiling for molecular classification of hereditary and sporadic forms of colorectal cancer. Their data show that miRNA expression profiling not only distinguished between tumor and normal tissues, but more importantly, a subset of miRNAs successfully discriminated among Lynch syndrome, sporadic microsatelliteunstable, and microsatellite-stable colorectal cancers. These findings could have tremendous clinical and translational relevance in the diagnosis, prognosis, and treatment strategy for patients with different subtypes of colorectal cancer.

Dual HER2-targeted approaches in HER2-positive breast cancer

Approximately 15-20% of all breast cancers are human epidermal growth factor receptor 2 (HER2) positive, with clinical studies having validated the HER2 receptor tyrosine kinase pathway as an important therapeutic target. Presently, two HER2-targeted therapies are approved by the Food and Drug Administration for treatment of HER2-positive breast cancer: the HER2-targeted humanized monoclonal antibody trastuzumab and the small-molecule tyrosine kinase inhibitor lapatinib. Despite use of these HER2-targeted agents, many patients still experience disease progression. For this reason, numerous new agents and therapeutic strategies are under investigation. Based on preclinical data suggesting synergistic effects from dual therapy targeting HER2, clinical trials that test the effects of combining anti-HER2 agents have been conducted and are ongoing. Here, we review recently presented data from several clinical trials, which indicate that the strategy of combining HER2 blockade therapies can offer greater clinical efficacy, with adverse effects of varying degrees. Specifically, we review new data reported at the 2010 San Antonio Breast Cancer Symposium (SABCS 2010), including the phase II NeoSphere and phase III NeoALTTO clinical trials, and data from three clinical trials reported at the 2011 American Society of Clinical Oncology (ASCO 2011) meeting. Together these trials elucidate the potential role of combining trastuzumab with lapatinib or pertuzumab. We also discuss additional ongoing studies that will help further define the role of dual HER2 blockade therapies and its impact on clinical practice.

What controls PTEN and what it controls (in prostate cancer)

What controls PTEN and what it controls (in prostate cancer)

Characterization of Novel Murine Monoclonal Antibodies Directed Against the Extracellular Domain of Human HER2 Tyrosine Kinase Receptor

HER2 proto-oncogene encodes a transmembrane receptor tyrosine kinase overexpressed in a variety of solid tumors. Several mouse monoclonal antibodies (MAbs) have been developed that recognize the extracellular part of HER2; of them two MAbs were humanized and employed for targeted immunotherapy. In this study we aimed to produce murine MAbs that specifically recognize the extracellular domain of human HER2. BALB/c mice were first primed with HER2-transfected NIH-3T3 cells and then boosted with recombinant extracellular part of HER2. Splenocytes from hyperimmunized mice were fused with myeloma cells and growing hybridomas were selected and screened for HER2 reactivity by an indirect ELISA. HER2-specific hybridomas were selected, cloned by limiting dilution assay, and further characterized by Western blotting and flow cytometry techniques. All clones showed positive reactivity to HER2 with binding affinity, ranging from 1.9×10(8) to 5×10(9), and stained HER2-transfected cells and malignant cells overexpressing HER2. None of the MAbs inhibited the binding of trastuzumab (Herceptin(®)) to HER2, indicating recognition of distinct epitopes by these MAbs. Based on these findings, our MAbs could be potentially used for selective targeting of HER2-expressing malignancies.

Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Abstract 233: Crosstalk of Her2 signaling and cell cycle control in the response to targeted therapeutics in breast cancer

Abstract Previous studies have shown that breast cancer is associated with genetic and molecular modifications, such as aberrant amplification of oncogenes and/or inactivation of tumor suppressors. This heterogeneity translates into a wide range of breast cancer subtypes that exhibit different disease behaviors and therapeutic sensitivity. A common oncogenic activation used to classify subtypes of breast cancer is the overexpression of human epidermal growth factor receptor 2 (Her2). These highly aggressive tumors present distinct characteristics in disease progression and therapeutic response. One of the main complications with current therapies is therapeutic bypass and disease recurrence. Therefore, a better understanding of the mechanisms of therapy resistance and the development of alternative downstream interventions is of utmost significance. There are several signaling cascades involved in tumor development that can be activated by the Her2 receptor tyrosine kinases. These signals ultimately converge on the cell cycle machinery, leading to the functional inactivation of RB to promote aberrant cell cycle progression and cell growth. In this context, RB pathway inactivation has been associated with aggressive tumor behavior and poor prognosis in breast cancer. In the current study, we explore the impact of RB inactivation in Her2 overexpressing disease in the response to Her2/EGFR antagonists. Furthermore, we assess the efficacy of reactivating the RB-pathway as a downstream therapeutic alternative in the treatment of Her2 overexpressing tumors and therapy-resistant disease in vitro and in vivo. In vitro studies demonstrate that RB loss can modulate the response to a Her2/EGFR antagonist. Alternatively, results indicate that a novel therapeutic agent that specifically targets cyclin dependent kinase 4 and 6 (CDK4/6) activity dramatically and efficiently inhibits acini/cell growth. Furthermore, CDK4/6 inhibition prevents the formation of multiacinar structures characteristic of Her2 overexpression. Furthermore, a Her2 overexpressing mouse mammary tumor model was utilized to confirm our in vitro findings. These studies indicate that CDK4/6 inhibition was effective in suppressing tumor cell proliferation in vivo. Combined, these findings provide further insight into the crosstalk between Her2 signaling and cell cycle control in the context of therapeutic response, and introduce a novel mode of therapeutic intervention. 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 233. doi:10.1158/1538-7445.AM2011-233

Abstract 1674: Significant reduction of primary growth and metastases through combined CXCR4 and HER-2 inhibition in an esophageal carcinoma orthotopic model

Abstract Metastatic disease is the main cause of morbidity and mortality in esophageal carcinoma and the leading cause of death. While in the early stages surgical resection is the best therapeutic option, in late stages the conventional therapies surgery, chemotherapy, and radiation are limited. The expression of the chemokine receptor CXCR4 is known to be involved in metastatic homing and is associated with a poor prognosis in esophageal cancer patients. Trastuzumab treatment leads to significant tumor growth reduction in a model of HER-2 positive esophageal carcinoma. In the present report, we show the effects of the humanized monoclonal antibody trastuzumab, the CXCR4 inhibitor AMD3100 and their combined use in a metastastic orthotopic nude mouse model of esophageal cancer. Although both single therapies lead to tumor growth reduction the effect is further enhanced by their combination and leads to significant reduction of metastases. Tumor progression was monitored by non invasive MR imaging. High correlation between HER-2 and CXCR4 expression in primary tumor and metastatic organs was observed and might suggest a link between the chemokine receptor CXCR4 and receptor tyrosine kinase HER-2 in tumor progression and metastases. The results of this study suggest an effective treatment of metastatic prostate cancer by the combined use of drugs that target HER-2 and CXCR4. 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 1674. doi:10.1158/1538-7445.AM2011-1674

Abstract 1616: The OPCML tumor suppressor negatively regulates a specific repertoire of 5 receptor tyrosine kinases via a novel proteasomal mechanism, and its recombinant derivative is a potent in-vivo anticancer protein therapy

Abstract OPCML, a GPI anchored tumor suppressor gene is inactivated by somatic methylation in multiple cancers. We previously identified this gene by LOH mapping and demonstrated that it was inactivated by somatic methylation in 80% of ovarian cancers. Restoring OPCML expression by stable transfection suppressed in-vitro growth and in-vivo tumorigenicity. We hypothesized that OPCML might abrogate growth signaling pathways. In SKOV-3 and PEO1, ovarian cancer cell lines with no expression of OPCML, we demonstrated that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1, FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA knockdown in normal ovarian surface epithelial cells up-regulates these same RTKs, with no effect on RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 and VEGFR3. Example immunoprecipitation experiments revealed that OPCML binds to EphA2, FGFR1 and HER2 extracellular domains with no such interaction to EGFR, thus OPCML binds directly to RTKS that it negatively regulates. We demonstrate that OPCML is located exclusively in the raft membrane fraction and sequesters RTKs that it binds to the raft fraction, leading to polyubiquitination and proteosomal degradation via a cav-1 endosomal mechanism resulting in systems depletion of this specific RTK repertoire, that does not occur with RTKs that OPCML does not bind. We demonstrate that OPCML abrogates EGF mediated phosphorylation of FGFR1, HER2 and EGFR and the downstream phosphosignaling of pErk and pAKT. A recombinant modified OPCML-like protein without a GPI anchor, signal peptide or glycosylation was constructed and expressed in E.Coli. This rOPCML tumor suppressor protein therapeutic caused growth inhibition by apoptosis in 6/7 ovarian cancer cell lines tested with no effect on OPCML expressing normal ovarian surface epithelium by an identical mechanism to the transfected normal protein. rOPCML was then injected intraperitoneally twice weekly in two murine intraperitoneal models of ovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profound inhibition of tumour weight, ascites volume and peritoneal dissemination compared with BSA control. In Summary, the OPCML tumour suppressor mediates its suppressor function by systems level negative regulation of at least 5 RTKs and a recombinant modified derivative is a potent tumor suppressor protein therapeutic in-vitro and in-vivo. 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 1616. doi:10.1158/1538-7445.AM2011-1616

Abstract 708: Integrated analysis of drug resistance networks in breast cancer

Abstract Targeted inhibition of HER2 tyrosine kinase receptor presents a promising strategy in the treatment of HER2-positive breast cancer patients. However, despite favorable response during the acute phase, majority of patients develop resistance to anti-HER2 drugs during the chronic phase of treatment. Here, we developed a cell culture model of chronic lapatinib (a dual inhibitor of EGFR and ERBB2 receptor tyrosine kinases) treatment by continuous passaging of HER2-overexpressing SKBR3 cells in increasing doses of lapatinib over a year. The resultant cell line, SKBR3-R, is almost 100-fold more resistant to lapatinib-induced growth inhibition when compared to the parental line. We have verified that this difference in lapatinib sensitivity is not due to altered expression or signaling at the level of HER2 or HER3 receptors in SKBR3-R cells. By integrating genome-wide gene expression profiling with our novel network-based genomic data analysis approach, we show that 1) cell toxicity mediated by acute lapatinib treatment in HER2-positive cells is associated with nutrient starvation and energy stress, and 2) chronic lapatinib treatment leads to the activation of compensatory cellular networks involved in the response to nutrient starvation, which confers resistance to lapatinib-induced cell death. Accordingly, while imposing a glucose starvation response phenotype on SKBR3 cells protects from lapatinib-mediated cell death, pharmacological inhibition of the nutrient starvation response in SKBR3-R cells reverses lapatinib resistance. In order to see if these networks play a role in breast cancer pathogenicity within clinical settings, we carried out a detailed network-based correlations of tumor gene expression profiles of breast cancer patients with their clinical data. Importantly, we found that the metabolic networks associated with lapatinib resistance are also associated with poor outcome within clinical settings. This study implicates nutrient deprivation response as a major factor in the acquired resistance of breast cancer cells to lapatinib, and suggests novel targets for combinatorial therapy of HER2-positive breast cancers. 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 708. doi:10.1158/1538-7445.AM2011-708