PI3K-independent mTOR activation promotes lapatinib resistance and IAP expression that can be effectively reversed by mTOR and Hsp90 inhibition

Although HER2 targeted therapies have substantially improved outcomes in HER2 overexpressing (HER2+) breast cancer, resistance to these therapies remains a clinical challenge. To better understand the mechanisms of resistance to lapatinib, a HER2 and EGFR dual kinase inhibitor, we treated HER2+ breast cancer cells with lapatinib for an extended period to generate a lapatinib-resistant (LapR) cell line model and examined cancer-promoting signaling activation in LapR cells. We found that LapR cells possess enhanced mTOR activation, which was independent of PI3K and other known mTOR activators. Lapatinib resistance could be reversed by mTOR kinase inhibition. Intriguingly, LapR cells had constitutive cytosolic cytochrome C, indicating that LapR cells suppress lapatinib-induced apoptosis downstream of cytochrome C release from mitochondria into the cytosol rather than by preventing its release into the cytosol. Consistent with this notion, LapR cells possessed increased levels of 2 of the inhibitors of apoptosis (IAPs), survivin and c-IAP-2, which are reported to block caspase activation downstream of cytosolic cytochrome C release. Further, treatment with the mTOR kinase inhibitor AZD8055 or the Hsp90 inhibitor 17-AAG reversed expression of IAPs and overcame lapatinib resistance in LapR cells. Together, these data suggest that suppression of apoptosis downstream of cytosolic cytochrome C release, possibly through increased expression of IAPs or other caspase-suppressing proteins, may promote lapatinib resistance. Further, PI3K is thought to be the main driver of lapatinib resistance, but our findings indicate that PI3K inhibitors may be ineffective in some lapatinib-resistant HER2+ breast cancers with PI3K-independent activation of mTOR kinase, which may instead benefit from mTOR or Hsp90 inhibitors.     

Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells

Introduction

Despite multiple advances in the treatment of HER2+ breast cancers, resistance develops even to combinations of HER2 targeting agents. Inhibition of PI3K pathway signaling is critical for the efficacy of HER2 inhibitors. Activating mutations in PIK3CA can overlap with HER2 amplification and have been shown to confer resistance to HER2 inhibitors in preclinical studies.

Methods

Lapatinib-resistant cells were profiled for mutations in the PI3K pathway with the SNaPshot assay. Hotspot PIK3CA mutations were retrovirally transduced into HER2-amplified cells. The impact of PIK3CA mutations on the effect of HER2 and PI3K inhibitors was assayed by immunoblot, proliferation and apoptosis assays. Uncoupling of PI3K signaling from HER2 was investigated by ELISA for phosphoproteins in the HER2-PI3K signaling cascade. The combination of HER2 inhibitors with PI3K inhibition was studied in HER2-amplified xenograft models with wild-type or mutant PIK3CA.

Results

Here we describe the acquisition of a hotspot PIK3CA mutation in cells selected for resistance to the HER2 tyrosine kinase inhibitor lapatinib. We also show that the gain of function conferred by these PIK3CA mutations partially uncouples PI3K signaling from the HER2 receptor upstream. Drug resistance conferred by this uncoupling was overcome by blockade of PI3K with the pan-p110 inhibitor BKM120. In mice bearing HER2-amplified wild-type PIK3CA xenografts, dual HER2 targeting with trastuzumab and lapatinib resulted in tumor regression. The addition of a PI3K inhibitor further improved tumor regression and decreased tumor relapse after discontinuation of treatment. In a PIK3CA-mutant HER2+ xenograft, PI3K inhibition with BKM120 in combination with lapatinib and trastuzumab was required to achieve tumor regression.

Conclusion

These results suggest that the combination of PI3K inhibition with dual HER2 blockade is necessary to circumvent the resistance to HER2 inhibitors conferred by PIK3CA mutation and also provides benefit to HER2+ tumors with wild-type PIK3CA tumors.     

Heregulin induces resistance to lapatinib‐mediated growth inhibition of HER2‐amplified cancer cells

Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 20% of gastric and gastroesophageal junction cancers in the United States and European Union. Lapatinib, a dual HER2 and epidermal growth factor receptor tyrosine kinase inhibitor, has demonstrated clinical efficacy in HER2‐amplified cancer cells. However, several studies have shown that some cytokines can mediate resistance to lapatinib using their receptor tyrosine kinase (RTK) pathways. One of these, Heregulin1 (HRG1), can confer resistance to lapatinib‐mediated growth inhibition in HER2‐amplified breast cancer cells, but the underlying mechanisms remain unknown. Here, we investigated whether and how HRG1 causes resistance to lapatinib in gastric and gastroesophageal junction cancers in vitro. HER2‐amplified gastric and gastroesophageal junction cancer cell lines were highly sensitive to lapatinib. Exposure to HRG1 together with lapatinib rescued cells from lapatinib‐induced cell cycle arrest and apoptosis. Downregulation of HER3 with siRNA in the presence of HRG1 re‐sensitized HER2‐amplified cancer cells to lapatinib. Immunoblotting analysis indicated that HRG1 re‐activated HER3 and AKT in the presence of lapatinib, which persisted for at least 72 h. Activation of HER3 and downstream AKT was mediated by residual activity of HER2. HRG1‐mediated resistance could be reduced by PI3K/mTOR inhibitors or by complete inhibition of HER2. Thus, we conclude that HRG1 mediates resistance to lapatinib through HER3 and AKT activation, and that this depends on residual HER2 activity. Lapatinib in combination with anti‐PI3K therapies or more potent HER2 inhibitors would improve the efficacy and avoid the emergence of resistant cells.     

乳腺癌 PIK3CA 基因突变与抗 HER2靶向治疗疗效的相关性

HER2阳性乳腺癌 PIK3CA 基因突变率高达25％,该基因突变能激活 PI3K-Akt 信号通路并促进 HER2介导的肿瘤细胞上皮转化,改变 HER2过表达肿瘤的内在表型,进而导致抗 HER2靶向治疗耐药。相关研究发现,PIK3CA 基因突变与抗 HER2靶向治疗疗效相关。因此,实时监测 PIK3CA 基因突变状态将有助于实现个体化抗 HER2靶向治疗。     

An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.     

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.     

BIM expression in treatment-naive cancers predicts responsiveness to kinase inhibitors

Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-na?ve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms.     

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.     

Combined effects of lapatinib and bortezomib in human epidermal receptor 2 (HER2)‐overexpressing breast cancer cells and activity of bortezomib against lapatinib‐resistant breast cancer cells

Lapatinib and bortezomib are highly active against breast cancer cells. Breast cancer patients who initially respond to lapatinib may eventually manifest acquired resistance to this treatment. Thus, the identification of novel agents that may prevent or delay the development of acquired resistance to lapatinib is critical. In the current study, we show that the combination of lapatinib and bortezomib results in a synergistic growth inhibition in human epidermal receptor 2 (HER2)‐overexpressing breast cancer cells and that the combination enhances apoptosis of SK‐BR‐3 cells. Importantly, we found that the combination of lapatinib plus bortezomib more effectively blocked activation of the HER2 pathway in SK‐BR‐3 cells, compared with monotherapy. In addition, we established a model of acquired resistance to lapatinib by chronically challenging SK‐BR‐3 breast cancer cells with increasing concentrations of lapatinib. Here, we showed that bortezomib notably induced apoptosis of lapatinib‐resistant SK‐BR‐3 pools and further inhibited HER2 signaling in the resistant cells. Taken together, the current data indicate a synergistic interaction between lapatinib and bortezomib in HER2‐overexpressing breast cancer cells and provide the rationale for the clinical evaluation of these two noncross‐resistant targeted therapies. The combination of lapatinib and bortezomib may be a potentially novel approach to prevent or delay the onset of acquired resistance to lapatinib in HER2‐overxpressing/estrogen receptor (ER)‐negative breast cancers. (Cancer Sci 2010); 00: 000–000     

RON confers lapatinib resistance in HER2-positive breast cancer cells

Lapatinib-resistance is a major problem for HER2-positive breast cancer treatment. SK-BR-3-LR, a lapatinib-resistant cell clone, was established from HER2-positive SK-BR-3 breast cancer cells following chronic exposure to lapatinib. The PI3K/AKT signaling pathway was demonstrated to be resistant to HER2 inhibition in SK-BR-3-LR cells. However, both small-molecular Recepteur d’Origine Nantais (RON) inhibitors and RON-targeted small interfering RNA (siRNA) effectively restored lapatinib sensitivity in these cells by inhibiting PI3K/AKT activation. Our results demonstrate for the first time the important role of RON in mediating lapatinib resistance and suggest that RON-targeted therapy may become a novel, promising therapeutic strategy after the failure of lapatinib treatment in patients with HER2-positive breast cancer.     

Expression of CD24 is associated with HER2 expression and supports HER2‐Akt signaling in HER2‐positive breast cancer cells

Human epidermal growth factor receptor 2 (HER2)‐positive breast cancer is treated with HER2‐targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3‐kinase (PI3K)‐Akt and MAPK pathways. However, resistance to HER2‐targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2‐positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple‐negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24‐positive cells was markedly higher in the HER2‐positive fraction than in the HER2‐negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2‐positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K‐Akt signaling. Furthermore, CD24 may contribute to resistance to HER2‐targeted therapy and, therefore, is itself a potential therapeutic target in HER2‐positive breast cancer.     

Berberine reverses lapatinib resistance of HER2-positive breast cancer cells by increasing the level of ROS

Lapatinib, a novel tyrosine kinase inhibitor of HER2/EGFR, is used to treat HER2-positive breast cancer. However, acquired drug resistance has limited the clinical therapeutic efficacy of lapatinib. Our previous study found that inhibition of autophagy can reduce the proliferation, DNA synthesis, and colony-forming capacity of lapatinib-resistant cells. Berberine has attracted extensive attention due to its wide range of biochemical and pharmacological effects in breast cancer treatment. It has been reported that berberine can induce oxidative stress and the mitochondrial-related apoptotic pathway in human breast cancer cells. In our current study, we found that a new combination therapy of berberine with lapatinib overcame lapatinib resistance. Furthermore, we found that berberine induced apoptosis of lapatinib-resistant cells through upregulating the level of ROS. Specially, lapatinib activated both the c-Myc/pro-Nrf2 pathway and GSK-3β signaling to stabilize Nrf2 and maintain a low level of ROS in resistant cells. However, berberine can upset the ROS balance by downregulating c-Myc to reverse the lapatinib resistance. Our finding provides a novel strategy of using berberine to overcome lapatinib resistance.     

Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.     

PIK3CA mutations and EGFR overexpression predict for lithium sensitivity in human breast epithelial cells

A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA "hotspot" mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN -/- cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors.     

Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable, whereas active phosphoinositide-3 kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFKs) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.     

Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

Introduction

The human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in breast cancer. Heterodimerization of HER2 with other HER family members results in enhanced tyrosine phosphorylation and activation of signal transduction pathways. HER2 overexpression increases the translation of fatty acid synthase (FASN), and FASN overexpression markedly increases HER2 signaling, which results in enhanced cell growth. However, the molecular mechanism and regulation of HER2 and FASN interaction are not well defined. Lapatinib is a small-molecule tyrosine kinase inhibitor that blocks phosphorylation of the epidermal growth factor receptor and HER2 in breast cancer cells, resulting in apoptosis. We hypothesized that FASN is directly phosphorylated by HER2, resulting in enhanced signaling and tumor progression in breast cancer cells.

Methods

Using mass spectrometry, we identified FASN as one of the proteins that is dephosphorylated by lapatinib in SKBR3 breast cancer cells. Immunofluorescence, immunoprecipitation, Western blotting, a kinase assay, a FASN enzymatic activity assay, an invasion assay, a cell viability assay and zymography were used to determine the role of FASN phosphorylation in invasion of SKBR3 and BT474 cells. The FASN inhibitor C75 and small interfering RNA were used to downregulate FASN expression and/or activity.

Results

Our data demonstrated that FASN is phosphorylated when it is in complex with HER2. FASN phosphorylation was induced by heregulin in HER2-overexpressing SKBR3 and BT474 breast cancer cells. Heregulin-induced FASN phosphorylation resulted in increased FASN enzymatic activity, which was inhibited by lapatinib. The FASN inhibitor C75 suppressed FASN activity by directly inhibiting HER2 and FASN phosphorylation. Blocking FASN phosphorylation and activity by lapatinib or C75 suppressed the activity of matrix metallopeptidase 9 and inhibited invasion of SKBR3 and BT474 cells.

Conclusions

FASN phosphorylation by HER2 plays an important role in breast cancer progression and may be a novel therapeutic target in HER2-overexpressing breast cancer cells.     

Promises borne out in clinical studies

This article reviews lapatinib clinical trials in patients with HER2 (ErbB2)-positive breast cancer, and is a report of a presentation from a symposium at the ECCO 14 congress in 2007.Promising clinical results have been achieved to date with lapatinib, an oral, intracellular, dual-targeted small molecule inhibitor of EGFR (ErbB1) and HER2 in patients with HER2-positive metastatic breast cancer. Lapatinib has shown impressive activity in HER2-positive metastatic breast cancer, both first-line and in heavily pretreated patients whose disease has progressed following prior treatment in the metastatic setting with taxanes, anthracyclines and trastuzumab. Lapatinib has also demonstrated activity in inflammatory breast cancer, a particularly aggressive form of the disease. Lapatinib is generally well tolerated, with the most common adverse events being diarrhoea and rash, which can be effectively managed with proactive guidelines.In conclusion, these data demonstrate that lapatinib is a promising new agent in the fight against HER2-positive breast cancer.     

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。