Simultaneous targeting of EGFR and mTOR inhibits the growth of colorectal carcinoma cells

Epidermal growth factor receptor (EGFR) is highly expressed in colorectal carcinomas and, as a result, it leads to the activation of downstream mammalian target of rapamycin (mTOR) kinase pathways for cancer growth and progression. Clinical and preclinical studies, however, have shown that inhibition of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) alone is not sufficient to treat colorectal carcinomas. In search of effective combination therapies, we show here that simultaneous targeting of EGFR with its inhibitor, erlotinib and mTOR with its inhibitor, rapamycin inhibits the phosphorylation and activation of downstream phosphatidylinositol 3-kinase (PI3K), Akt, mTOR and extracellular-signal-regulated kinase 1/2 (Erk1/2) pathways, resulting in the inhibition of cell cycle progression and the growth of both KRAS wild-type and mutated colorectal carcinoma cells. This study has demonstrated the principle that the combination of erlotinib and rapamycin may provide an effective therapy for colorectal carcinomas.     

Involvement of the Akt/mTOR pathway on EGF-induced cell transformation

Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.     

The mammalian target of rapamycin pathway is widely activated without PTEN deletion in renal cell carcinoma metastases

BACKGROUND:

Inhibitors of the mammalian target of rapamycin (mTOR) are emerging as promising therapies for metastatic renal cell carcinoma (RCC). Because rational treatment strategies require understanding the activation status of the underlying signaling pathway being targeted at the desired stage of disease, the authors examined the activation status of different components of the mTOR pathway in RCC metastases and matched primary tumors.

METHODS:

The authors immunostained metastatic RCC samples from 132 patients and a subset of 25 matched primary RCCs with antibodies against phosphatidylinositol 3′‐kinase, PTEN, phospho‐Akt, phospho‐mTOR, and p70S6. PTEN genomic status was assessed by fluorescent in situ hybridization. Marker expression was correlated to clinicopathologic variables and to survival.

RESULTS:

The mTOR pathway showed widespread activation in RCC metastases of various sites with strong correlation between different components of this signaling cascade (P<.0001), but without significant PTEN genomic deletion. Only cytoplasmic phospho‐mTOR showed independent prognostic significance (P = .029) and fidelity between primary RCCs and their matched metastases (P = .004).

CONCLUSIONS:

Activation of various components of the mTOR signaling pathway in metastatic RCC lesions across various tumor histologies, nuclear grades, and metastatic sites suggests the potential for vertical blockade of multiple steps of this pathway. Patient selection may be improved by mTOR immunostaining of primary RCC. Cancer 2011. © 2010 American Cancer Society.     

Activation of mammalian target of rapamycin pathway confers adverse outcome in nonsmall cell lung carcinoma

BACKGROUND:

Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been shown to contribute to tumorigenesis. This study explored protein expression profiles of mTOR pathway and the relationship with prognosis in patients with nonsmall cell lung carcinoma (NSCLC).

METHODS:

The protein expression profiles of mTOR/phosphorylated (p‐)mTOR, phosphoinositide‐dependent kinase 1 (PDK1)/p‐PDK1, p‐Akt1, and P70 ribosomal protein S6 kinase (P70S6K)/p‐P70S6K were determined via immunohistochemical staining assay. The clinical prognostic values of both single and combined protein expression were investigated with univariate and multivariate survival analysis.

RESULTS:

Compared with normal lung tissues, the protein levels of mTOR/p‐mTOR, p‐Akt1 Ser473/Thr308, and P70S6K/p‐P70S6K were higher (all P < .05), whereas p‐PDK1 was lower (P < .05) in tumor tissues. p‐mTOR expression was associated with histological differentiation, histological type, lymph node invasion, and stage (all P < .05). Overall survival in NSCLC patients was significantly shorter in cases with positive phenotype for p‐mTOR, p‐PDK1, and p‐P70S6K (all P < .05). Subjects with coexpression of any 2 of p‐mTOR, p‐PDK1, p‐Akt1 Ser473, and p‐P70S6K demonstrated worse prognosis than those expressing no biomarker or any 1 biomarker alone (all P < .05). Multivariate analysis showed that the combination of p‐mTOR/p‐P70S6K is an independent prognostic factor in addition to tumor stage.

CONCLUSIONS:

This study provides clinical evidence that activated components of mTOR pathway, not total protein, are predictors of poor prognosis in NSCLC. Moreover, evaluating protein‐expression profiles of these molecules might be a new strategy for individual therapy in subjects with NSCLC. Cancer 2011;. © 2011 American Cancer Society.     

Comparison of Akt/mTOR signaling in primary breast tumors and matched distant metastases

BACKGROUND.

The Akt/mammalian target of the rapamycin (mTOR) signaling pathway represents a promising target for cancer therapy. The phosphorylation status of Akt and of mTOR's phosphorylation target eukaryotic initiation factor 4E‐binding protein 1 (4E‐BP1) is often used to assess the activity of Akt and mTOR signaling. The purpose of this study was to determine whether primary tumors differ from their metastasis in their expression of pAkt and p4E‐BP1.

METHODS.

Primary breast tumors and their distant metastases surgically resected from the same patients were evaluated with immunohistochemical analysis (IHC) for pAkt (Ser473) and p4E‐BP1 (Ser65). The agreement between the IHC results for the primary tumor and metastases was evaluated with Cohen kappa (κ).

RESULTS.

Most primary breast tumors and metastatic tumors expressed pAkt (76% of each). Of the 23 matched evaluable pairs, however, 11 (47.8%) had discordant IHC results (κ ?0.31; 95% confidence interval [CI], ?0.49 to ?0.13). Similarly, although most of the primary and metastatic tumors were positive for p4E‐BP1 (75% and 74%), of the 23 matched evaluable pairs, 8 (47.8%) were discordant (κ 0.10; 95% CI, ?0.33–0.52).

CONCLUSIONS.

In this series, most primary breast tumors and metastases expressed pAkt and p4E‐BP1 by IHC. Concordance between IHC findings in primary tumors and metastases was poor, however. Further work is needed to determine whether this reflects true biological heterogeneity or poor reproducibility of IHC with phosphospecific antibodies, and to identify which biomarkers can be assessed most reproducibly in primary tumors to predict activity of Akt/mTOR signaling and sensitivity to pathway inhibitors. Cancer 2008. ©2008 American Cancer Society.     

Collagen type I induces EGFR‐TKI resistance in EGFR‐mutated cancer cells by mTOR activation through Akt‐independent pathway

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR‐TKI sensitivity in EGFR‐mutant cells. We evaluated the EGFR‐TKI sensitivity of EGFR‐mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR‐TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR‐TKI treatment. In cancer cells cultured with and without Col I, EGFR‐TKI suppressed the levels of phosphorylated (p‐)EGFR, p‐ERK1/2, and p‐Akt. When compared to cancer cells without Col I, expression of p‐P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR‐TKI treatment. Simultaneous treatment with EGFR‐TKI and mTOR inhibitor abrogated Col I‐induced resistance to EGFR‐TKI. Patients with Col I‐rich stroma had a significantly shorter progression‐free survival time after EGFR‐TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt‐independent pathway, which results in EGFR‐TKI resistance. Combination therapy using EGFR‐TKI and mTOR inhibitor could be a possible strategy to combat this resistance.     

Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways

The Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, differentiation, and survival. However, the role of the Akt/mTOR/p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC) cell growth and survival. Herein, we explore the role of the Akt/mTOR/p70S6K pathway in fibronectin-induced NSCLC cell growth. We found that fibronectin stimulated the phosphorylation of Akt, an upstream inducer of mTOR, and induced the phosphorylation of p70S6K1 and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of mTOR in NSCLC cells (H1792 and H1838), whereas it inhibited the phosphatase and tensin homologue deleted on chromosome 10, a tumor suppressor protein that antagonizes the phosphatidylinositol 3-kinase/Akt signal. In addition, treatment with fibronectin inhibited the mRNA and protein expression of LKB1 as well as the phosphorylation of AMP-activated protein kinase (AMPKalpha), both known to down-regulate mTOR. Rapamycin, an inhibitor of mTOR, blocked the fibronectin-induced phosphorylation of p70S6K and 4E-BP1. Akt small interfering RNA (siRNA) and an antibody against the fibronectin-binding integrin alpha5beta1 also blocked the p70S6K phosphorylation in response to fibronectin. In contrast, an inhibitor of extracellular signal-regulated kinase 1/2 (PD98095) had no effect on fibronectin-induced phosphorylation of p70S6K. Moreover, the combination of rapamycin and siRNA for Akt blocked fibronectin-induced cell proliferation. Taken together, these observations suggest that fibronectin-induced stimulation of NSCLC cell proliferation requires activation of the Akt/mTOR/p70S6K pathway and is associated with inhibition of LKB1/AMPK signaling.     

Loss of tuberous sclerosis complex-2 function and activation of mammalian target of rapamycin signaling in endometrial carcinoma.

PURPOSE: The involvement of phosphatase and tensin homologue deleted on chromosome ten (PTEN) in endometrial carcinoma has implicated phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin (mTOR) activation in this disease. Understanding the extent of mTOR involvement and the mechanism responsible for activation is important, as mTOR inhibitors are currently being evaluated in clinical trials for endometrial carcinoma. Although tuberous sclerosis complex 2 (TSC2) is the "gatekeeper" for mTOR activation, little is known about defects in the TSC2 tumor suppressor or signaling pathways that regulate TSC2, such as LKB1/AMP-activated protein kinase, in the development of endometrial carcinoma. EXPERIMENTAL DESIGN: We determined the frequency of mTOR activation in endometrial carcinoma (primary tumors and cell lines) and investigated PTEN, LKB1, and TSC2 defects as underlying cause(s) of mTOR activation, and determined the ability of rapamycin to reverse these signaling defects in endometrial carcinoma cells. RESULTS: Activation of mTOR was a consistent feature in endometrial carcinomas and cell lines. In addition to PTEN, loss of TSC2 and LKB1 expression occurred in a significant fraction of primary tumors (13% and 21%, respectively). In tumors that retained TSC2 expression, phosphorylation of tuberin at S939 was observed with a high frequency, indicating that mTOR repression by TSC2 had been relieved via AKT phosphorylation of this tumor suppressor. In PTEN-null and LKB1-null endometrial carcinoma cell lines with functional inactivation of TSC2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 were able to inhibit AKT and mTOR signaling and reverse TSC2 phosphorylation. In contrast, although rapamycin inhibited mTOR signaling, it did not relieve phosphorylation of TSC2 at S939. CONCLUSIONS: Inactivation of TSC2 via loss of expression or phosphorylation occurred frequently in endometrial carcinoma to activate mTOR signaling. High-frequency mTOR activation supports mTOR as a rational therapeutic target for endometrial carcinoma. However, whereas rapamycin and its analogues may be efficacious at inhibiting mTOR activity, these drugs do not reverse the functional inactivation of TSC2 that occurs in these tumors.     

雷帕霉素靶蛋白信号通路在食管癌细胞系中激活状态的研究

[目的]研究雷帕霉素靶蛋白（mTOR）信号传导通路在食管鳞癌细胞系EC9706和Eca109中的激活状态。[方法]采用细胞免疫组化检测mTOR及其下游靶分子（p70S6K）在两细胞系中的表达．并采用RT-PCR和Western blot方法分别从mRNA水平及蛋白水平测定此通路的活性。[结果]mTOR在两种细胞系中均有表达，且在EC9706中的表达水平明显高于Eca109，mTOR下游靶点（p706S6K）的磷酸化水平也是EC9706明显高。[结论]mTOR信号通路在两种细胞系中都是特异性激活。激活状态与细胞的分化程度有关，细胞分化程度越低，通路的激活水平越高。     

PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation

Background:

Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.

Methods:

Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.

Results:

Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.

Conclusion:

Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.     

Dissecting the Akt/mammalian target of rapamycin signaling network: emerging results from the head and neck cancer tissue array initiative.

PURPOSE: As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from head and neck squamous cell carcinoma (HNSCC) patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high-density HNSCC tissue microarray, with a high representation of oral squamous cell carcinoma. EXPERIMENTAL DESIGN: These tissue arrays were constructed by acquiring cylindrical biopsies from multiple individual tumor tissues and transferring them into tissue microarray blocks. From a total of 1,300 cases, 547 cores, including controls, were selected and used to build the array. RESULTS: Emerging information by the use of phosphospecific antibodies detecting the activated state of signaling molecules indicates that the Akt-mammalian target of rapamycin (mTOR) pathway is frequently activated in HNSCC, but independently from the activation of epidermal growth factor receptor or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of epidermal growth factor receptor activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR. CONCLUSIONS: These findings provide important information about the nature of the dysregulated signaling networks in HNSCC and may also provide the rationale for the future development of novel mechanism-based therapies for HNSCC patients.     

mTOR siRNA对喉鳞状细胞癌Hep-2细胞生物学行为的影响

目的观察mTOR siRNA对喉鳞状细胞癌Hep-2细胞增殖能力的影响。方法选取45例喉鳞状细胞癌组织和45例正常喉黏膜组织,采用免疫组织化学法检测mTOR蛋白的表达;将喉鳞状细胞癌Hep-2细胞分为空白对照组、空载体组和mTOR siRNA组,及正常对照组、溶剂对照组和雷帕霉素组,分别利用mTOR siRNA和雷帕霉素进行治疗,采用Western bloting法检测不同浓度的mTOR siRNA和雷帕霉素分别处理细胞后对mTOR蛋白表达的影响,并采用CCK-8试剂盒分析mTOR蛋白表达下调对喉鳞状细胞癌Hep-2细胞增殖能力的影响。结果 mTOR蛋白在喉鳞状细胞癌组织中表达的阳性率显著高于正常喉黏膜组织(P<0.05)。3种不同浓度mTOR siRNA分别转染Hep-2细胞24、48、72 h,各组与空白对照组、空载体组相比,mTOR蛋白表达均有所降低(P<0.05),且mTOR蛋白表达随mTOR siRNA浓度的增加、作用时间的延长而降低(P<0.05)。3种不同浓度雷帕霉素处理Hep-2细胞24、48、72 h,各组与溶剂对照组及正常对照组相比,mTOR蛋白表达量均有所降低(P<0.05);且mTOR蛋白表达随雷帕霉素浓度的增加、处理时间的延长而降低(P<0.05)。与空白对照组、空载体组相比,各mTOR siRNA组的Hep-2细胞增殖在转染后24、48、72 h均受到明显抑制(P<0.05)。结论 mTOR siRNA均能降低喉鳞状细胞癌Hep-2细胞中mTOR蛋白的表达,有效抑制细胞的生长,为喉鳞状细胞癌的分子治疗提供了理论依据。     

mTOR通路及其抑制剂抗肿瘤的研究进展

哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是PI3K/Akt通路的下游分子,可接受生长因子、营养、能量等多种信号,是细胞生长和增殖的关键调节分子.许多肿瘤中存在有编码mTOR信号通路相关蛋白的基因突变,这些蛋白的异常表达可引起mTOR通路的过度激活.目前,以mTOR为治疗靶点成为肿瘤治疗的研究新热点.本文就近年来有关mTOR通路及其抑制剂在抗肿瘤方面的研究进展作一综述.     

P63 differentiates subtypes of nonsmall cell carcinomas of lung in cytologic samples

BACKGROUND:

Bevacizumab in combination with carboplatin and paclitaxel improves overall response and survival in patients with advanced or recurrent nonsmall cell lung carcinoma. However, this drug is not recommended in patients with squamous cell carcinoma or neoplasms with a dominant squamous component. Therefore, identification of squamous cell differentiation has therapeutic implications. In many instances, cytology is the diagnostic tool of choice; however, routine cytomorphology is limited in classification of nonsmall cell carcinomas into squamous and nonsquamous subtypes. The aim of this study was to identify the value of p63 immunocytochemical analysis in this distinction.

METHODS:

Review of cytology records identified 51 consecutive pulmonary specimens (16 fine needle aspiration samples, 15 washes, 12 brushes, and 8 lavages) with the diagnosis of nonsmall cell lung carcinoma (9 carcinomas with squamous differentiation and 42 carcinomas without squamous differentiation). Histologically, they all proved to be nonsmall cell carcinomas, 26 with squamous differentiation and 25 without squamous differentiation. p63 immunocytochemical stain was performed on archival alcohol‐fixed Papanicolaou‐stained cytology slides using standard immunocytochemical methods.

RESULTS:

Twenty‐three (88 %) of the 26 histologically proven squamous cell carcinomas were positive for p63 on cytologic smears. By using p63 immunocytochemistry, the authors detected 14 carcinomas with squamous differentiation not identified by cytomorphology. Smears from all histologically proven carcinomas with squamous differentiation were positive for p63. Sensitivity of cytology for the detection of nonsmall cell carcinoma of lung with squamous differentiation increased from 35% to 88% using p63 immunocytochemistry (P = .001; McNemar test). The squamous component in 4 carcinomas was detected only in cytologic and not in corresponding histologic samples when subsequent p63 immunostaining was performed.

CONCLUSIONS:

The authors concluded that p63 is a useful marker for the detection of nonsmall cell carcinomas of lung with squamous differentiation when used in cytologic pulmonary samples. p63 immunocytochemistry significantly increases the sensitivity for the identification of lung neoplasms with squamous differentiation from 35% to 88% (P = .001). Therefore, p63 immunocytochemistry may be used in pulmonary cytologic samples of nonsmall cell carcinomas to identify squamous differentiation and to improve therapeutic selection of patients with lung cancer. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Mutational Damages in Malignant Lung Tumors

Background: Today, genomic changes are an important cause of the occurrence, growth and progression of cancer. Technological advances in cancer genomic analysis platforms have made it possible to identify genomic alterations that may influence response to lung cancer treatment. Methods: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. Results: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. HRAS mutations were present in most squamous cell carcinomas and adenocarcinomas of the lung. EcoR1- and Pst1- restriction enzymes destroyed the RT-PCR product of the p53 and p21Waf1 mRNA and increased the level of detected mutations in lung adenocarcinoma to 75% and 50 %, respectively. EGFR mutations were more frequent in lung adenocarcinoma than in lung squamous cell carcinoma. Mutations in EGFR exons 19 and 21 found in 65 of 263 lung tumor samples indicated the tumor sensitivity to EGFR tyrosine kinase inhibitors. EGFR deletions in exon 19 occurred mainly in adenocarcinoma, L858R mutations in EGFR exon 21 were quite common in lung adenocarcinoma. Conclusion: The mutations detected in most squamous cell carcinomas and adenocarcinomas of the lung could be used to diagnose and predict the disease severity and targeted therapy efficacy.     

The novel phosphoinositide 3‐kinase–mammalian target of rapamycin inhibitor,BEZ235, circumvents erlotinib resistance of epidermal growth factor receptor mutant lung cancer cells triggered by hepatocyte growth factor

Acquired resistance to epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs), such as gefitinib and erlotinib, is a critical problem in the management of patients with EGFR mutant lung cancer. Several mechanisms have been reported involved in this acquired resistance, including hepatocyte growth factor (HGF) activation of an alternative pathway. PI3K and mTOR are downstream molecules of receptor tyrosine kinases, such as EGFR and Met, and are thought to be ideal targets for controlling various tumor types. We assessed whether BEZ235, a dual inhibitor of PI3K and mTOR, could overcome the EGFR–TKI resistance induced by HGF in an EGFR mutant lung cancer model. Exogenous and endogenous HGF triggered resistance to erlotinib in the PC‐9 and HCC827, EGFR mutant lung cancer cell lines. BEZ235 alone inhibited the viability of PC‐9 and HCC827 cells in vitro, irrespective of the presence or the absence of HGF. Using a xenograft model of severe combined immunodeficient mice with HGF‐gene‐transfected PC‐9 cells (PC‐9/HGF), we found that BEZ235 inhibited tumor growth, whereas erlotinib did not. BEZ235 monotherapy also inhibited the phosphorylation of Akt and p70S6K/S6RP, downstream molecules of PI3K and mTOR, respectively, as well as suppressing tumor‐cell proliferation and angiogenesis of PC‐9/HGF tumors. These results suggest that BEZ235, even as monotherapy, may be useful in managing HGF‐induced EGFR–TKI resistance in EGFR mutant lung cancer.     

Overexpressed eIF4E is functionally active in surgical margins of head and neck cancer patients via activation of the Akt/mammalian target of rapamycin pathway.

PURPOSE: Overexpression of eIF4E in surgical margins of head and neck cancer patients is an independent risk factor for recurrence. We hypothesize that overexpressed eIF4E is functionally active in tumor margins through activation of the Akt/mammalian target of rapamycin (mTOR) pathway EXPERIMENTAL DESIGN: Western blots and/or immunohistochemistry were performed to determine whether phosphorylation of mTOR and activation of its downstream molecules eIF4E-binding protein-1 (4E-BP1) and p70 S6 kinase and the upstream modulator of mTOR, Akt, were expressed in margins overexpressing eIF4E. RESULTS: There was a significant association between phospho-4E-BP1 and eIF4E expression of a margin or a significant difference in phospho-4E-BP1 expression between the eIF4E-positive and -negative margins (P < 0.01). A significant association between eIF4E and phospho-p70 S6 kinase as well as eIF4E and phospho-mTOR was also noted (P < 0.05). Western blot analysis indicated a highly significant difference in the phosphorylation status of 4E-BP1 between tumors and resection margins. A total of 89% of the 4E-BP1-expressing margins expressed more of the phosphorylated (beta, gamma, and delta) isoforms, whereas 81% of the 4E-BP1-expressing tumors expressed more of the unphosphorylated alpha isoform. A similar difference in Akt activation was noted between eIF4E-positive margins and tumors (P < 0.05). CONCLUSIONS: Overexpression of eIF4E is functionally active in tumor margins through activation of the Akt/mTOR signaling pathway. The greater degree of expression of downstream targets and upstream regulators of mTOR in margins compared with the tumors indicates preferential activation of the Akt/mTOR signaling pathway in margins overexpressing eIF4E. Rapamycin analogs can potentially be used as adjuvant therapy for patients with eIF4E-positive margins.     

Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors.

PURPOSE: Human and murine preneoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the Akt/mammalian target of rapamycin (mTOR) pathway, suggesting a role for this pathway in lung cancer development. To test this, we did studies with rapamycin, an inhibitor of mTOR, in A/J mice that had been exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). EXPERIMENTAL DESIGN: Tumorigenesis was induced by i.p. injection of NNK, and rapamycin was administered 1 or 26 weeks after NNK administration. Biomarkers associated with mTOR inhibition were assessed in lung and/or surrogate tissues using immunohistochemistry and immunoblotting. Rapamycin levels were measured using mass spectroscopy. RESULTS: Rapamycin was administered on a daily (5 of 7 days) regimen beginning 26 weeks after NNK decreased tumor size, proliferative rate, and mTOR activity. Multiplicity was not affected. Comparing this regimen with an every-other-day (qod) regimen revealed that rapamycin levels were better maintained with qod administration, reaching a nadir of 16.4 ng/mL, a level relevant in humans. When begun 1 week after NNK, this regimen was well tolerated and decreased tumor multiplicity by 90%. Tumors that did develop showed decreased phenotypic progression and a 74% decrease in size that correlated with decreased proliferation and inhibition of mTOR. CONCLUSIONS: Tobacco carcinogen-induced lung tumors in A/J mice are dependent upon mTOR activity because rapamycin markedly reduced the development and growth of tumors. Combined with the Food and Drug Administration approval of rapamycin and broad clinical experience, these studies provide a rationale to assess rapamycin in trials with smokers at high risk to develop lung cancer.