Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY‐142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines

Mutations in KRAS or BRAF frequently manifest in constitutive activation of the MEK1/2‐ERK1/2 signalling pathway. The MEK1/2‐selective inhibitor, AZD6244 (ARRY‐142886), blocks ERK1/2 activation and is currently undergoing clinical evaluation. Tumour cells can vary markedly in their response to MAPK or ERK kinase (MEK) inhibitors, and the presence of a BRAF mutation is thought to predict sensitivity, with the RAS mutations being associated with intrinsic resistance. We analysed cell proliferation in a panel of 19 colorectal cancer cell lines and found no simple correlation between BRAF or KRAS mutation and sensitivity to AZD6244, though cells that harbour neither mutation tended to be resistant. Cells that were sensitive arrested in G₁ and/or underwent apoptosis and the presence of BRAF or KRAS mutation was not sufficient to predict either fate. Cell lines that were resistant to AZD6244 exhibited low or no ERK1/2 activation or exhibited coincident activation of ERK1/2 and protein kinase B (PKB), the latter indicative of activation of the PI3K pathway. In cell lines with coincident ERK1/2 and PKB activation, sensitivity to AZD6244 could be re‐imposed by any of the 3 distinct PI3K/mTOR inhibitors. We conclude that AZD6244 is effective in colorectal cancer cell lines with BRAF or KRAS mutations. Sensitivity to MEK1/2 inhibition correlates with a biochemical signature; those cells with high ERK1/2 activity (whether mutant for BRAF or KRAS) evolve a dependency upon that pathway and tend to be sensitive to AZD6244 but this can be offset by high PI3K‐dependent signalling. This may have implications for the use of MEK inhibitors in combination with PI3K inhibitors. © 2009 UICC     

Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

Effect of a poly(ADP‐ribose) polymerase‐1 inhibitor against esophageal squamous cell carcinoma cell lines

Effective molecular target drugs that improve therapeutic efficacy with fewer adverse effects for esophageal cancer are highly anticipated. Poly(ADP‐ribose) polymerase (PARP) inhibitors have been proposed as low‐toxicity agents to treat double strand break (DSB)‐repair defective tumors. Several findings imply the potential relevance of DSB repair defects in the tumorigenesis of esophageal squamous cell carcinoma (ESCC). We evaluated the effect of a PARP Inhibitor (AZD2281) on the TE‐series ESCC cell lines. Of these eight cell lines, the clonogenic survival of one (TE‐6) was reduced by AZD2281 to the level of DSB repair‐defective Capan‐1 and HCC1937 cells. AZD2281‐induced DNA damage was implied by increases in γ‐H2AX and cell cycle arrest at G2/M phase. The impairment of DSB repair in TE‐6 cells was suggested by a sustained increase in γ‐H2AX levels and the tail moment calculated from a neutral comet assay after X‐ray irradiation. Because the formation of nuclear DSB repair protein foci was impaired in TE‐6 cells, whole‐exome sequencing of these cells was performed to explore the gene mutations that might be responsible. A novel mutation in RNF8, an E3 ligase targeting γ‐H2AX was identified. Consistent with this, polyubiquitination of γ‐H2AX after irradiation was impaired in TE‐6 cells. Thus, AZD2281 induced growth retardation of the DSB repair‐impaired TE‐6 cells. Interestingly, a strong correlation between basal expression levels of γ‐H2AX and sensitivity to AZD2281was observed in the TE‐series cells (R² = 0.5345). Because the assessment of basal DSB status could serve as a biomarker for selecting PARP inhibitor‐tractable tumors, further investigation is warranted.     

AZD6244 (ARRY‐142886) enhances the antitumor activity of rapamycin in mouse models of human hepatocellular carcinoma

BACKGROUND:

The protein kinase B (AKT)/mammalian target of rapamycin (AKT/mTOR) and mitogen activated protein kinase/extracellular regulated kinase kinase/extracellular regulated kinase (MEK/ERK) signaling pathways have been shown to play an important role in hepatocellular carcinoma (HCC) growth and angiogenesis, suggesting that inhibition of these pathways may have therapeutic potential.

METHODS:

We treated patient‐derived HCC xenografts with 1) mTOR inhibitor rapamycin (RAPA); 2) MEK inhibitor AZD6244 (ARRY‐142886); and 3) AZD6244 plus RAPA (AZD6244/RAPA). Western blotting was used to determine pharmacodynamic changes in biomarkers relevant to angiogenesis, mTOR pathway, and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry.

RESULTS:

We report here that pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the antitumor and antiangiogenic activities of mTOR inhibitor RAPA in both orthotopic and ectopic models of HCC. Such inhibition led to increased apoptosis, decreased angiogenesis and cell proliferation, reduced expression of positive cell cycle regulators, and increase in proapoptotic protein Bim.

CONCLUSIONS:

Our findings indicate that the AZD6244/RAPA combination had antitumor and antiangiogenic effects in preclinical models of human HCC. Given the urgent need for effective therapies in HCC, clinical evaluating AZD6244/RAPA combination seems warranted. Cancer 2010. © 2010 American Cancer Society.     

Micro‐RNA‐21 regulates TGF‐β‐induced myofibroblast differentiation by targeting PDCD4 in tumor‐stroma interaction

Transforming growth factor‐β1 (TGF‐β1) induces stromal fibroblast‐to‐myofibroblast transdifferentiation in the tumor‐stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro‐RNAs (miRNAs) and their roles in TGF‐β1‐induced myofibroblast differentiation in tumor‐stroma interaction are unclear. Using quantitative real‐time RT‐PCR, we demonstrated that the expression of micro‐RNA‐21 (miR‐21) was upregulated in activated fibroblasts after treatment with TGF‐β1 or conditioned medium from cancer cells. To determine the potential roles of miR‐21 in TGF‐β1‐mediated gene regulation during myofibroblast conversion, we showed that miR‐21 expression was downregulated by miR‐21 inhibitor and upregulated by miR‐21 mimic. Interestingly, downregulation of miR‐21 with the inhibitor effectively inhibited TGF‐β1‐induced myofibroblast differentiation while upregulation of miR‐21 with a mimic significantly promoted myofibroblast differentiation. We further demonstrated that MiR‐21 directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myofibroblasts. Taken together, these results suggested that miR‐21 participated in TGF‐β1‐induced myofibroblast transdifferentiation in cancer stroma by targeting PDCD4.     

BRAF V600E disrupts AZD6244-induced abrogation of negative feedback pathways between extracellular signal-regulated kinase and Raf proteins

AZD6244 (ARRY 142886) is a potent and selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor currently in early clinical trials. We examined the activity of AZD6244 in a panel of non-small cell lung cancer and a panel of cell lines representing many cancer types using in vitro growth assays. AZD6244 induced G(0)-G(1) cell cycle arrest in sensitive cell lines that primarily included cells containing the BRAF V600E mutation. In these cells, G(0)-G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1) and down-regulation of cyclin D1. In the majority of cell lines tested, including those with K-ras or non-V600E BRAF mutations, AZD6244 induced the accumulation of phospho-MEK, an effect not observed in the most sensitive BRAF V600E-containing cells. Accumulation of phospho-MEK in non-V600E-containing cell lines is due to abrogation of negative feedback pathways. BRAF V600E disrupts negative feedback signaling, which results in enhanced baseline phospho-MEK expression. Exogenous expression of BRAF V600E disrupts feedback inhibition but does not sensitize cells to AZD6244. Specific suppression of endogenous BRAF V600E does not confer resistance to AZD6244 but enhances sensitivity to AZD6244. Thus, our findings show that BRAF V600E marks cells with an in vitro requirement for MAPK signaling to support proliferation. These cells are exquisitely sensitive to AZD6244 (IC(50), <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition between ERK and Raf. These data suggest an approach to identifying cells that may be sensitive to AZD6244 and other MEK inhibitors.     

Inhibition of mammalian target of rapamycin signaling potentiates the effects of all‐trans retinoic acid to induce growth arrest and differentiation of human acute myelogenous leukemia cells

Our study explored the drug interaction of all‐trans retinoic acid (ATRA) and RAD001 (everolimus), the inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in acute myelogenous leukemia (AML) NB4 and HL60 cells. RAD001 (10 nM) significantly enhanced the ATRA‐induced growth arrest and differentiation of these cells, as measured by colony‐forming assay and cell cycle analysis, and expression of CD11b cell surface antigen and nitroblue tetrazolium reduction, respectively. ATRA (0.1–1 μM) upregulated levels of RTP801, a negative regulator of mTORC1, and inhibited mTORC1 signaling as assessed by measurement of the levels of p‐p70S6K and p‐4E‐BP1 in HL60 and NB4 cells. ATRA (0.1–1 μM) in combination with RAD001 (10 nM) strikingly downregulated the levels of p‐70S6K and p‐4E‐BP1 without affecting the total amount of these proteins. Notably, RAD001 (10 nM) significantly augmented ATRA‐induced expression of CCAAT/enhancer‐binding protein ε (C/EBPε) and p27^kip1 and downregulated levels of c‐Myc in these cells. Furthermore, RAD001 (5 mg/kg) enhanced the ability of ATRA (10 mg/kg) to inhibit the proliferation of HL60 cells growing as tumor xenografts in immune‐deficient nude mice. Taken together, concomitant blockade of the RA and mTORC1 signaling may be a promising treatment strategy for individuals with AML. © 2009 UICC     

Combination of the ERK inhibitor AZD6244 and low-dose sorafenib in a xenograft model of human renal cell carcinoma

Sorafenib, a multikinase inhibitor, is currently used as monotherapy for advanced renal cell carcinoma (RCC). However, adverse effects associated with its use have been experienced by some patients. In this study, we examined the antitumor and antiangiogenic activities of low-dose sorafenib in combination with the MEK inhibitor AZD6244 (sorafenib/AZD6244) in a preclinical model of RCC. Primary RCC 08-0910 and RCC?786-0 cells as well as patient-derived RCC models were used to study the antitumor and antiangiogenic activities of sorafenib/AZD6244. Changes of biomarkers relevant to angiogenesis and cell cycle were determined by western immunoblotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of RCC 786-0 cells with sorafenib/AZD6244 resulted in G1 cell cycle arrest and blockade of serum-induced cell migration. Sorafenib/AZD6244 induced apoptosis in primary RCC 08-0910 cells at low concentrations. In?vivo addition of AZD6244 to sorafenib significantly augmented the antitumor activity of sorafenib and allowed dose reduction of sorafenib without compromising its antitumor activity. Sorafenib/AZD6244 potently inhibited angiogenesis and phosphorylation of VEGFR-2, PDGFR-β and ERK, p90RSK, p70S6K, cdk-2 and retinoblastoma. Sorafenib/AZD6244 also caused upregulation of p27, Bad and Bim but downregulation of survivin and cyclin B1. These resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. Our findings showed that AZD6244 and sorafenib complement each other to inhibit tumor growth. This study provides sound evidence for the clinical investigation of low-dose sorafenib in combination with AZD6244 in patients with advanced RCC.     

Down-regulation of mitogen-inducible gene 6, a negative regulator of EGFR, enhances resistance to MEK inhibition in KRAS mutant cancer cells

Previously, we found that KRAS mutant cancer cells showed variable response to AZD6244, a MEK inhibitor through differential activation of EGFR/AKT. To investigate its mechanism, we performed cDNA microarray using four KRAS mutant cancer cells. We found that treatment with AZD6244 reduced the expression of mitogen-inducible gene 6 (MIG6), a negative feedback regulator for EGFR, in AZD6244-resistant cells, while activity of EGFR and AKT was increased in these cells. Reconstitution or knockdown of MIG6 expression affected cancer cell responses to AZD6244. Treatment with a combination of EGFR inhibitor and AZD6244 inhibited cell proliferation synergistically without activation of AKT in AZD6244-resistant cells. Our study provides a mechanism of differential response to MEK inhibition in KRAS mutant cancer.     

N6‐isopentenyladenosine inhibits cell proliferation and induces apoptosis in a human colon cancer cell line DLD1

N⁶‐isopentenyladenosine (i6A) is a modified nucleoside with a pentaatomic isopentenyl derived from mevalonate that induces inhibition of tumor cell proliferation and apoptosis in several tumor cell lines. In this study, we reported that N⁶‐isopentenyladenosine inhibited the proliferation and promotes apoptosis in DLD1 human colon cancer cells. It suppressed the proliferation of cells through inhibition of DNA synthesis, causing a cell cycle arrest that correlated with a decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a concomitant increase in the levels of cyclin‐dependent kinase inhibitor p21waf and p27kip1. Moreover, it induced apoptosis through an increase in the number of annexin V‐positive cells, a downregulation of antiapoptotic products and caspase‐3 activation. The apoptotic effects of N⁶‐isopentenyladenosine were accompanied by sustained phosphorylation and activation of c‐jun N‐terminal kinase (JNK) that induced phosphorylation of c‐jun. Overall, our data show that JNK, could play an important role in i6A‐mediated apoptosis in DLD1 human colon cancer cells © 2008 Wiley‐Liss, Inc.     

Blockade of MEK/ERK signaling enhances sunitinib-induced growth inhibition and apoptosis of leukemia cells possessing activating mutations of the FLT3 gene

The FMS-like tyrosine kinase 3 (FLT3) is a cell surface receptor tyrosine kinase. Activating mutations of this gene occur in nearly 30% of acute myelogenous leukemia (AML) patients. These mutations, in part, result in activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. In this study, we found that AZD6244 (ARRY-142886), a novel inhibitor of MEK1/2 kinases, effectively inhibited the proliferation of acute biphenotypic leukemia MV4-11 and acute monocytic leukemia MOLM13 cells. The concentrations that inhibited 50% growth were approximately 0.3 and 1.2 microM, respectively, as measured by thymidine uptake on day 2 of culture. AZD6244 potently down-regulated the levels of phospho-ERK1/2 and its downstream effector, p-p70S6K, in the MV4-11 and MOLM13 cells as measured by Western blot analysis. Interestingly, when AZD6244 was combined with sunitinib, a FLT3 kinase inhibitor, growth inhibition and apoptosis of both MV4-11 and MOLM13 cells were synergistically enhanced in association with further down-regulation of phospho-ERK1/2 and p-p70S6K in these cells. Taken together, concomitant blockade of FLT3 and MEK signaling represents a promising treatment strategy for individuals with leukemia who possess activating mutations of FLT3.     

Inhibition of class I histone deacetylases by romidepsin potently induces Epstein‐Barr virus lytic cycle and mediates enhanced cell death with ganciclovir

Pan‐histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein‐Barr virus (EBV) lytic cycle in EBV‐associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV‐associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC‐1, ‐2 and ‐3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA‐approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ～0.5 to 5 nM (versus ～800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21^WAF1, which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21^WAF1 and induction of lytic cycle were abrogated by a specific inhibitor of PKC‐δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC‐1, ?2 and ?3 by romidepsin or shRNA knockdown could confer susceptibility of EBV‐positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV‐associated cancers.     

Hypoxia‐inducible factor‐1α and nuclear factor‐κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non‐small‐cell lung cancer cells

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.     

Protease‐activated receptor‐2 accelerates intestinal tumor formation through activation of nuclear factor‐κB signaling and tumor angiogenesis in ApcMin/+ mice

Hepatocyte growth factor activator inhibitor‐1 (HAI‐1), encoded by the SPINT1 gene, is a membrane‐bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor‐1 regulates type II transmembrane serine proteases that activate protease‐activated receptor‐2 (PAR‐2). We previously reported that deletion of Spint1 in Apc^Min/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor‐κB signaling. In this study, we examined the role of PAR‐2 in accelerating tumor formation in the Apc^Min/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR‐2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI‐1 deficiency was also normalized by compound deficiency of PAR‐2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in Apc^Min/+‐induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR‐2, and that HAI‐1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR‐2 activating proteases.     

ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

Extracellular adenosine 5′‐triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of β‐catenin as well as its downstream target genes CD44, c‐Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP‐driven events in vitro and in vivo. Furthermore, iCRT14, a β‐catenin/TCF complex inhibitor, could also suppress ATP‐driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2‐β‐catenin axis. Thus blockade of the ATP‐P2Y2‐β‐catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.