The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines

HER2 overexpression is observed in 5-25% of gastric cancers. Lapatinib is a dual inhibitor of the epidermal growth factor receptor and HER2 tyrosine kinase. We examined the antitumor effect of lapatinib in gastric cancer cell lines. Lapatinib induced selective and potent growth inhibition in two HER2-amplified gastric cancer cell lines (SNU-216 and NCI-N87). Lapatinib inhibited the phosphorylation of HER2, EGFR and downstream signaling proteins, resulting in G1 arrest in both cell lines with down-regulation of cMyc and induction of p27kip1. Lapatinib also induced apoptosis in NCI-N87 which has high HER2 amplification ratio. Lapatinib combined with 5-fluorouracil, cisplatin, oxaliplatin or paclitaxel showed an additive or synergistic effect. These results provide a rationale for the future clinical trials of lapatinib combined with cytotoxic drugs in the treatment of HER2-positive gastric cancer.     

Lapatinib acts on gastric cancer through both antiproliferative function and augmentation of trastuzumab-mediated antibody-dependent cellular cytotoxicity

Background

Trastuzumab has been recently approved for clinical use to treat HER2-expressing advanced gastric cancer, and anti-HER2-targeting therapy has become a promising option for gastric cancer. Lapatinib is a dual tyrosine kinase inhibitor targeting EGFR and HER2. The aim of the present study was to explore the utility of lapatinib for gastric cancer, with a particular focus on trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC).

Methods

Nine gastric cancer cell lines were evaluated for the effects of lapatinib on the cell-surface accumulation of HER2 and analyzed for their additional effects on trastuzumab-mediated ADCC. Also, HER2 signaling with Western blot, proliferative function with the MTT assay, and apoptosis-inducing activity with 7ADD/Annexin-V were investigated when a panel of gastric cancer cell lines was treated with lapatinib.

Results

Lapatinib inhibited HER2 signaling and cell proliferation in the panel of gastric cancer cell lines. Lapatinib also induced the accumulation of HER2 on the cell surface, resulting in the enhancement of trastuzumab-mediated ADCC of gastric cancer.

Conclusions

Lapatinib exhibits inhibitory activity in gastric cancer cells, and the combination of lapatinib with trastuzumab may be a promising treatment strategy for gastric cancer patients.     

Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo.

Aberrrant signaling by the epidermal growth factor receptor [EGFR (HER1, erbB1)] and/or HER2/neu tyrosine kinases is present in a cohort of breast carcinomas. Because HER2 is constitutively phosphorylated in some breast tumors, we speculated that, in these cancers, transmodulation of HER2 may occur via EGFR signaling. To test this possibility, we examined the effect of EGFR-specific kinase inhibitors against the HER2-overexpressing human breast tumor lines BT-474, SKBR-3, MDA-361, and MDA-453. ZD1839 (Iressa) is an ATP-mimetic that inhibits the purified EGFR and HER2 kinases in vitro with an IC(50) of 0.033 and >3.7 microM, respectively. The specificity of ZD1839 against EGFR was confirmed in Rat1 fibroblasts transfected with EGFR or HER2 chimeric receptors activated by synthetic ligands without the interference of endogenous receptors. Treatment of all breast cancer cell lines (except MDA-453) with 1 microM ZD1839 almost completely eliminated HER2 phosphorylation. In contrast, the incorporation of [gamma-(32)P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 microM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene.     

The effect of immune-based therapy with cytotoxic T lymphocyte and molecular targeting therapy for HER2 in esophageal squamous cell carcinoma

Although esophageal squamous cell carcinoma (ESCC) patients have recently been treated with the combined modality therapy, the prognosis remains poor. For the development of new strategies in ESCC, we examined possibilities of the immune -based therapy with cytotoxic T lymphocyte (CTL) and the molecular targeting therapy for HER2 against ESCC in this study. At first, we assessed HER2 and MHC class I expression by immunohistochemistry in ESCC patients and analyzed the correlation between them. Subsequently, the effect of molecular targeting therapy for HER2 was evaluated in a panel of ESCC cell lines. According to these results, we suggested that HER2 over-expressing ESCC patients (11.8%) are good candidates for the molecular targeting therapy for HER2 and HER2 negative/low-expressing ESCC patients (88.2%) for the immune-based therapy with CTL. Furthermore, the combination therapy of Herceptin and lapatinib is a new promising strategy for HER2 positive ESCC patients (29.4%).     

Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.     

Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported.Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab.Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α.EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma.     

拉帕替尼联合顺铂抗食管鳞癌的作用及机制

目的 探讨拉帕替尼联合顺铂抗食管鳞癌的体外活性及其作用机制。方法 MTT法检测拉帕替尼、顺铂单独及二者联合对食管鳞癌细胞的增殖抑制作用，并计算两药联合作用指数。碘化丙啶（PI）染色或Annexin V-FITC/PI双染结合流式细胞术检测细胞周期进程及凋亡，Western blot法检测两药单独及联合处理后食管鳞癌细胞EGFR和HER2的磷酸化及下游信号分子的表达变化。结果 拉帕替尼联合顺铂可协同抑制食管鳞癌细胞的增殖，二者之间的联合作用指数小于1。拉帕替尼联合顺铂可使食管鳞癌细胞阻滞于G2/M期，并可显著诱导细胞凋亡。联合用药可显著抑制EGFR和HER2的磷酸化，并进而抑制两个主要的下游信号分子ERK和AKT的活化。结论 拉帕替尼联合顺铂在体外具有协同抗食管鳞癌活性，对EGFR和HER2过表达食管鳞癌是一种有前景的治疗策略。     

Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells

Background:

Cervical cancer (CC) annually kills 288 000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximab''s toxicity plus chemoradiation on CC cells, which express different EGFR levels.

Methods:

EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations.

Results:

Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximab''s cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

Conclusion:

Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.     

Effects of the EGFR/HER2 kinase inhibitor GW572016 on EGFR- and HER2-overexpressing breast cancer cell line proliferation, radiosensitization, and resistance

PURPOSE: Two members of the epidermal growth factor receptor family, EGFR and HER2, have been implicated in radioresistance in breast cancer and other malignancies. To gauge the potential clinical utility of targeting both EGFR and HER2 to control growth and radiosensitize human breast cancers, we examined the effect of a dual EGFR/HER2 inhibitor, GW572016, on the proliferation and radiation response of either EGFR- or HER2-overexpressing human breast cancer cell lines. METHODS AND MATERIALS: Primary human breast cancer cell lines that endogenously overexpress EGFR or HER2 and luminal mammary epithelial H16N2 cells stably transfected with HER2 were evaluated for the effect of GW572016 on inhibition of ligand-induced or constitutive receptor phosphorylation, proliferation, radiosensitization, and inhibition of downstream signaling. RESULTS: GW572016 inhibited constitutive and/or ligand-induced EGFR or HER2 tyrosine phosphorylation of all five cell lines, which correlated with the antiproliferative response in all but one cell line. GW572016 radiosensitized EGFR-overexpressing cell lines, but HER2-overexpressing cells were unable to form colonies after brief exposure to GW572016 even in the absence of radiation, and thus could not be evaluated for radiosensitization. One cell line was resistant to the antiproliferative and radiosensitizing effects of GW572016, despite receptor inhibition. Exploration of potential mechanisms of resistance in SUM185 cells revealed failure of GW572016 to inhibit downstream ERK and Akt activation, despite inhibition of HER2 phosphorylation. In contrast, sensitive HER2-overexpressing cell lines demonstrated inhibition of both ERK and Akt phosphorylation. CONCLUSION: GW572016 potently inhibits receptor phosphorylation in either EGFR- or HER2-overexpressing cell lines and has both antiproliferative and radiosensitizing effects. Resistance to GW572016 was not due to a lack of receptor inhibition, but rather with a lack of inhibition of ERK and Akt, suggesting that measurement of inhibition of crucial signaling pathways may better predict response than inhibition of receptor phosphorylation. The SUM185 cell line provides a valuable model for studying mechanisms of resistance of EGFR/HER2 inhibitor therapy.     

In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2^low human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab′)₂ fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.     

Concurrent biological targeting therapy of squamous cell carcinoma of the esophagus with cetuximab and trastuzumab

The ERBB proteins are cell membrane tyrosine kinase receptors. Among these receptors, ERBB1 (EGFR or HER1) and ERBB2 (HER2/Neu) have been reported to be the most important in terms of the development and progression of squamous cell carcinoma of the esophagus (SCC). Thus, targeting of ERBB1 and ERBB2 may become a promising strategy to treat SCC. In the present study, we examined ERBB1 and ERBB2 expression of SCC cell lines (TT, TE2, TE6 and TE10) and tumor samples. In addition, we evaluated the effect of the anti-ERBB1 antibody cetuximab and the anti-ERBB2 antibody trastuzumab for SCC in vitro and in vivo. Biological activities (receptor downregulation, the phosphorylation of MAPK and AKT) induced by the two agents were also investigated. Immunohistochemistry of SCC samples showed that ERBB1 was detected in 84%, while ERBB2 was detected in 30%. RT-PCR analysis revealed that ERBB1 and ERBB2 mRNA were detectable in all four cell lines. MTT cell proliferation analysis showed that cetuximab, but not trastuzumab, inhibited growth in each of the SCC cell lines in a dose-dependent manner. Further, cetuximab and trastuzumab used together produced stronger inhibition of growth compared to cetuximab alone. Cetuximab downregulated ERBB1, but not ERBB2, at the TE6 cell surface. However, neither ERBB1 nor ERBB2 showed any downregulation by trastuzumab at the TE6 cell surface. Cetuximab, not but trastuzumab, inhibited the phosphorylation of MAPK and Akt. When administered in combination, the two agents inhibited Akt phosphorylation to a greater degree compared to treatment with cetuximab alone. In the in vivo study, cetuximab, but not trastuzumab, significantly inhibited the TT tumors. Additionally, the combination of cetuximab with trastuzumab induced a synergistic inhibitory antitumor effect in the TT tumors. In conclusion, combination of cetuximab and trastuzumab revealed a synergistic antitumor effect for SCC in vitro and in vivo. The antitumor effect may be induced by the inhibition of the phosphorylation of Akt. These findings suggest that combination therapy including cetuximab and trastuzumab may be a promising strategy to treat SCC.     

Lysophosphatidic acid-induced squamous cell carcinoma cell proliferation and motility involves epidermal growth factor receptor signal transactivation

Transactivation of the epidermal growth factor receptor (EGFR) represents the paradigm for cross-talk between G protein-coupled receptors (GPCRs) and receptor tyrosine kinase signaling pathways. In a variety of squamous cell carcinoma cell lines of the head and neck (HNSCCs), we found that treatment with the GPCR agonists lysophosphatidic acid (LPA), bradykinin, thrombin, and carbachol results in rapid tyrosine phosphorylation of the EGFR. In these tumor cells, signal transactivation of the EGFR and the oncoprotein HER2/neu is critically dependent on metalloprotease activity. Using the metalloprotease inhibitor batimastat, the EGFR-specific tyrphostin AG1478, and a dominant-negative EGFR mutant, we show that in HNSCC cell lines, EGFR tyrosine phosphorylation, recruitment of the adaptor proteins SHC and Gab1, and activation of the ERK/mitogen-activated protein kinase pathway in response to LPA depend both on metalloprotease function and EGFR tyrosine kinase activity. Most importantly, critical characteristics of HNSCC cell lines such as DNA synthesis, cell cycle progression and tumor cell migration are stimulated by LPA and can be abrogated by interfering with EGFR signal transmission. Together, our results demonstrate the importance of a mechanism that promotes head and neck cancer cell proliferation and motility by GPCR ligands involving EGFR transactivation. Our findings suggest that highly abundant GPCR ligands such as LPA may function as tumor promoters and determinants of HNSCC progression.     

Small cell lung cancer cells express EGFR and tyrosine phosphorylation of EGFR is inhibited by gefitinib ("Iressa", ZD1839)

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839) has demonstrated anti-tumor activity in non-small cell lung cancer (NSCLC) and has been approved in over 20 countries. NSCLC has been reported to express high levels of EGFR. However, gefitinib appears to be more effective against adenocarcinoma than squamous cell carcinoma, the latter expressing more EGFR. In the present study, we evaluated the effect of gefitinib against the small cell lung cancer (SCLC) cell lines NCI-H82, NCI-H209, NCI-H510, NCI-H526 and NCI-H660. SCLC has been reported to express a low to undetectable level of EGFR. We compared the effects of gefitinib between cell lines with detectable and undetectable EGFR expression. First, we evaluated expression levels of EGFR and HER2/neu by Western blotting and immunoprecipitation respectively; EGFR protein was detected in two of the five SCLC cell lines, whereas HER2/neu was not detected in any. Next, we analyzed expression levels of phosphorylated ERK1/2 and compared these results with EGFR (HER-1/ErbB1) and HER2/neu (ErbB2) expression levels, as EGFR conducts signals through Ras-Raf-MAPK pathway; gefitinib inhibited phosphorylation of ERK1/2 by EGF addition in cell lines with detectable and undetectable EGFR expression. These data suggest that gefitinib is potentially effective against cancers with low EGFR expression such as SCLC.     

Lapatinib resistance in HER2+ cancers: latest findings and new concepts on molecular mechanisms

In the era of new and mostly effective molecular targeted therapies, human epidermal growth factor receptor 2 positive (HER2+) cancers are still intractable diseases. Lapatinib, a dual epidermal growth factor receptor (EGFR) and HER2 tyrosine kinase inhibitor, has greatly improved breast cancer prognosis in recent years after the initial introduction of trastuzumab (Herceptin). However, clinical evidence indicates the existence of both primary unresponsiveness and secondary lapatinib resistance, which leads to the failure of this agent in HER2+ cancer patients. It remains a major clinical challenge to target the oncogenic pathways with drugs having low resistance. Multiple pathways are involved in the occurrence of lapatinib resistance, including the pathways of receptor tyrosine kinase, non-receptor tyrosine kinase, autophagy, apoptosis, microRNA, cancer stem cell, tumor metabolism, cell cycle, and heat shock protein. Moreover, understanding the relationship among these mechanisms may contribute to future tumor combination therapies. Therefore, it is of urgent necessity to elucidate the precise mechanisms of lapatinib resistance and improve the therapeutic use of this agent in clinic. The present review, in the hope of providing further scientific support for molecular targeted therapies in HER2+ cancers, discusses about the latest findings and new concepts on molecular mechanisms underlying lapatinib resistance.     

计算机辅助设计HER2/neu酪氨酸激酶小分子抑制剂及其生物活性研究

目的：用计算机辅助设计法寻找HER2/neu受体酪氨酸激酶小分子抑制剂。方法：用MODERLAR软件模拟出HER2/neu和EGFR受体酪氨酸激酶的三维(three dimensional,3D)空间结构;根据HER2/neu酪氨酸激酶ATP结合区的空间结构,搜索数据库,找出候选化合物;用Westernblot方法检测化合物对HER2/neu受体酪氨酸激酶磷酸化的影响;MTT法检测细胞增殖抑制作用。结果：比较HER2/neu和EGFR（靶蛋白）与胰岛素受体酪氨酸激酶、FGFR1受体酪氨酸激酶和Src酪氨酸激酶（模板蛋白）的氨基酸序列发现有35％～41％的相同和52％～55％的相似,用MODELLER程序模拟出HER2/neu和EGFR激酶区域的3D结构。根据HER2/neu受体酪氨酸激酶结构模型,3D数据库的搜索,获得潜在HER2/neu酪氨酸激酶抑制剂化合物,经筛选、优化发现ST2325对HER2/neu磷酸化有明显的抑制作用,其IC50值为6.6μmol/L,且抑制作用是可逆的,对EGFR受体磷酸化没有抑制作用。ST2325对HER2/neu受体表达没有影响。ST2325对HER2/neu过表达的肿瘤细胞MDA MB 453m1的抑制作用更强,而对EGFR过表达的肿瘤细胞MDA MB 468抑制作用相对较弱,其IC50值分别为29.05μmol/L和60.40μmol/L。结论：基于HER2/neu的结构设计,筛选出对HER2/neu酪氨酸激酶有明显选择性抑制作用的ST2325。     

The dual EGFR/HER‐2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN‐38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER‐2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN‐38, the active metabolite of irinotecan (CPT‐11), in colon and gastric cancer cell lines. Concentration‐dependent antiproliferative effects of both lapatinib and SN‐38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08–11.7 μM; SN‐38 range 3.6–256 nM). Lapatinib potently inhibited the growth of a HER‐2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER‐2 expression. The combination of lapatinib and SN‐38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN‐38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN‐38 when compared to SN‐38 treatment alone. Finally, the combination of lapatinib and CPT‐11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT‐11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas. © 2009 UICC     

Gene status of head and neck squamous cell carcinoma cell lines and cetuximab-mediated biological activities

Cetuximab is a chimeric IgG1 monoclonal antibody that targets epidermal growth factor receptor (EGFR). Cetuximab binds to EGFR and prevents phosphorylation of EGFR. Moreover, preclinical results have shown the ability of cetuximab to induce either complement-mediated tumor cell killing (CDC) or antibody-dependent cell-mediated-cytotoxicity (ADCC). We previously reported mutation in EGFR regarding head and neck squamous cell carcinoma (HNSCC) cell lines. In the present study, we analyzed the same 16 HNSCC cell lines for mutations in KRAS, PIK3CA, BRAF and PTEN. Furthermore, we evaluated cetuximab-mediated biological activities (antiproliferative effect by the MTT assay and ADCC) regarding these cell lines. Mutations in PIK3CA and PTEN were observed in two cell lines (2/16, 12.5%), but no mutation was observed in KRAS and BRAF. The antiproliferative effect of cetuximab by the MTT assay was not strong, and no correlation was observed between the antiproliferative effect of cetuximab and mutations in EGFR, KRAS, PIK3CA, BRAF and PTEN in these cell lines. Therefore, the mutation status of EGFR and downstream molecules were not useful for predicting the antitumor effects of cetuximab on HNSCC. Cetuximab-mediated ADCC was observed in these cell lines and might have been influenced by the expression of EGFR. Therefore, cetuximab-mediated ADCC seems to be an important part of the antitumor mechanisms of cetuximab and the expression levels of EGFR might influence the antitumor activity of cetuximab. Therefore, besides the antiproliferative effect of cetuximab by the MTT assay, it appeared important to evaluate cetuximab-mediated ADCC as well as EGFR expression in HNSCC cells.     

Non-small cell lung cancer clinical trials with trastuzumab: their foundation and preliminary results

The overexpression of HER2, a transmembrane glycoprotein tyrosine kinase, has been implicated in mitogenesis, cell survival, invasion and angiogenesis. Preclinical evidence suggests that HER2 overexpression contributes to tumor progression in non-small cell lung cancer (NSCLC) and retrospective clinical correlative studies show that it is probably associated with poor clinical outcome. Trastuzumab (Herceptin, Genentech Inc., South San Francisco, CA) is a recombinant humanized monoclonal antibody that targets HER2 and is currently approved for use in the treatment of patients with HER2-overexpressing metastatic breast cancer. Two primary mechanisms proposed for the activity of trastuzumab are downregulation of HER2 and induction of antibody-dependent cell-mediated cytotoxicity. Evidence from preclinical studies of trastuzumab in NSCLC and other cell lines, the presence of HER2 overexpression in NSCLC clinical specimens and the clinical benefit derived from trastuzumab in phase II and III metastatic breast cancer trials have led to the development of clinical trials of trastuzumab in NSCLC. Phase II studies of trastuzumab in patients with stage IIIB or IV NSCLC are being conducted to test the efficacy of trastuzumab as a single agent or in combination with chemotherapy. Preliminary results show combinations of chemotherapy plus trastuzumab are well tolerated, with encouraging response rates of 21-40%. A randomized phase II trial of chemotherapy with or without trastuzumab showed promise in a small subgroup of patients with 3+ HER2 overexpression by immunohistochemistry or HER2 DNA amplification by fluorescence in situ hybridization. Taken together, these data indicate that trastuzumab warrants further investigation in a clinical study in selected patients with NSCLC.     

Tyrosine phosphorylation of PYK2 mediates heregulin-induced glioma invasion: novel heregulin/HER3-stimulated signaling pathway in glioma

Receptor tyrosine kinases of the EGFR family transmit extracellular signals that control diverse cellular functions such as proliferation, differentiation and survival. Signaling function of a member of this family, HER3, is believed to be impaired due to deviations in its kinase consensus motifs. Here we address the functional role and signaling mechanisms of HER3. HER3 preferentially forms heterodimers with HER2 inducing the most potent mitogenic signal among EGFR family members. Our data show that in a glioma-derived cell line the cytoplasmic tyrosine kinase PYK2 is constitutively associated with HER3 and that stimulation with Heregulin results in PYK2 tyrosine phosphorylation. HER3, but not HER2, mediates the phosphorylation of the C-terminal region of PYK2 to promote a mitogenic response through activation of the MAPK pathway. A central role of PYK2 in signaling downstream of HER3 is substantiated by the demonstration that expression of a dominant-negative PYK2-KM construct abrogates the Heregulin-induced MAPK activity and inhibits the invasive potential of glioma cells. These results suggest a novel Heregulin/HER3-stimulated signaling pathway in glioblastoma-derived cell lines that involves phosphorylation of PYK2 and mediates invasiveness of glioma cells.