COX-2 inhibitors and genetic background reduce mammary tumorigenesis in cyclooxygenase-2 transgenic mice

Cyclooxygenase-2 (COX-2) overexpression is a widely recognized feature of human breast cancer and inhibitors of the enzyme have antitumor effects in a subset of tumor settings. Previously, we demonstrated that direct overexpression of COX-2 under control of the mammary-specific MMTV promoter/enhancer, was itself oncogenic and lead to the induction of mammary tumors in multiparous, outbred CD1 mice. In the present study, we provide evidence that COX-2 dependent tumor progression can also be studied in FVB/N, an inbred strain widely used for analysis of breast cancer progression. In these mice, the human COX-2 transgene was strongly induced during pregnancy/lactation and mammary tumors developed after multiple pregnancies. However, crossing the COX-2 FVB/N mice with the C57BL6 strain resulted in loss of the mammary tumorigenic phenotype despite the fact that the human COX-2 gene was induced. Treatment of the COX-2 transgenic mice in the FVB/N strain with celecoxib (1600 ppm), a COX-2 selective inhibitor, resulted significant reduction in tumor size and multiplicity when compared to transgenic mice fed with control chow. SC-560 (20 ppm), a COX-1 selective inhibitor did not have significant effect on tumorigenesis. These studies suggest that FVB/N is a susceptible mouse strain well suited to the study of COX-2 mediated tumor progression and may provide a tool for the identification of interacting genes and therapeutic treatments for this clinically important target.     

Nitric oxide signaling in colon cancer chemoprevention

Nitric oxide (NO) is a pleiotrophic regulator, pivotal to numerous biological processes, including vasodilation, neurotransmission, and macrophage-mediated immunity. The highly reactive free radicals, produced by NO synthases (NOS) have been implicated in the modulation of carcinogenesis. Over-expression of inducible NOS (iNOS), a common phenomenon during chronic inflammatory conditions, generates sustainable amounts of NO, that its reactive intermediates are mutagenic, causing DNA damage or impairment of DNA repair, has been well established in carcinogenesis. Recent studies also implicate NO as having a key signaling molecule that regulates processes of tumorigenesis. Increased expression of iNOS has been observed in tumors of the colon, lung, oropharynx, reproductive organs, breast, and central nervous system besides its occurrence in chronic inflammatory diseases. Progression of a large majority of human and experimental colon tumors appears to progress by NO resulting from stimulation of proinflammatory cytokines, and inactivation (nitrosylation) of p53 mediated caspase activities in the tumors, whereas in some cases it associated with induction of apoptosis and tumor regression. This dichotomy is largely explained by the complexity of signaling pathways in tumor cells, that respond to NO very differently depending on its concentration. p53 mutation, functional loss, activation, and inactivation of apoptotic proteins all have been linked with NO resistance and dependence. Evidence from both in vitro and in vivo experiments support that NO and its reactive metabolite peroxynitrite stimulate COX-2 activity leading generation of tumor growth enhancing prostaglandins. Thus, NO mediated signaling can augment the tumor growth and metastasis by promoting invasive and angiogenic properties of tumor cells, which includes triggering and activation of COX-2. Thus, developing selective inhibitors of iNOS and NO-releasing agents may lead to important strategies for chemoprevention of colon cancer. Chemoprevention studies at preclinical level with several selective inhibitors of iNOS in both chemically and transgenic models of colon cancer are encouraging.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Survivin and Cyclooxygenase-2 are co-expressed in human and mouse colon carcinoma and in terminally differentiated colonocytes

In the evolution of colon rectal cancer (CRC) the imbalance between cell proliferation and apoptosis is considered one of the prominent causes of tumor induction and/or progression. In order to establish the role of anti apoptotic proteins in colon cancer development, we studied with immunohistochemical techniques the expression of Survivin in a mouse model of colon carcinogenesis induced by 1,2-dimethyl-hydrazine treatment. In this mouse model Survivin was over-expressed during tumor development, showing a distribution mimicking that described in the correspondent human malignancies. We also correlated Survivin distribution with COX-2 and beta-Catenin expression patterns. The co-localization of COX-2/beta-Catenin/Survivin in the same epithelial cells in tumor samples lends credence to possible in vivo regulatory effects of COX-2 and beta-Catenin on the intracellular Survivin levels in mouse and human colon cancer.     

Potential role of microsomal prostaglandin E synthase-1 in tumorigenesis

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is a stimulus-inducible enzyme that functions downstream of cyclooxygenase (COX)-2 in the PGE2-biosynthetic pathway. Given the accumulating evidence that COX-2-derived PGE2 participates in the development of various tumors, including colorectal cancer, we herein examined the potential involvement of mPGES-1 in tumorigenesis. Immunohistochemical analyses demonstrated the expression of both COX-2 and mPGES-1 in human colon cancer tissues. HCA-7, a human colorectal adenocarcinoma cell line that displays COX-2- and PGE2-dependent proliferation, expressed both COX-2 and mPGES-1 constitutively. Treatment of HCA-7 cells with an mPGES-1 inhibitor or antisense oligonucleotide attenuated, whereas overexpression of mPGES-1 accelerated, PGE2 production and cell proliferation. Moreover, cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when implanted subcutaneously into nude mice. cDNA array analyses revealed that this mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to proliferation, morphology, adhesion, and the cell cycle. These results collectively suggest that aberrant expression of mPGES-1 in combination with COX-2 can contribute to tumorigenesis.     

环氧合酶-2 在肿瘤中的表达及其抑制剂在肿瘤中的应用进展

环氧合酶-2(Cyclooxygenase-2,COX-2)是前列腺素合成过程中一重要的限速酶,COX-2的过度表达及其前列腺素产物与多种肿瘤的发生、发展关系密切,COX-2抑制剂通过抑制肿瘤细胞增殖,诱导肿瘤细胞凋亡,阻断致癌物的代谢,减弱肿瘤介导的免疫抑制,调节抑制血管生成,抑制肿瘤细胞侵袭,环氧合酶非依赖抑癌途径,对原癌基因及抑癌基因的影响等途径影响肿瘤的发生发展,这方面的研究为针对COX-2的抗肿瘤策略打开新的视野,提供新的线索。     

The functional interaction between Acyl-CoA synthetase 4, 5-lipooxygenase and cyclooxygenase-2 controls tumor growth: a novel therapeutic target

The acyl-CoA synthetase 4 (ACSL4), which esterify mainly arachidonic acid (AA) into acyl-CoA, is increased in breast, colon and hepatocellular carcinoma. The transfection of MCF-7 cells with ACSL4 cDNA transforms the cells into a highly aggressive phenotype and controls both lipooxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) metabolism of AA, suggesting a causal role of ACSL4 in tumorigenesis. We hypothesized that ACSL4, LOX-5 and COX-2 may constitute potential therapeutic targets for the control of tumor growth. Therefore, the aim of this study was to use a tetracycline Tet-Off system of MCF-7 xenograft model of breast cancer to confirm the effect of ACSL4 overexpression on tumor growth in vivo. We also aim to determine whether a combinatorial inhibition of the ACSL4-LOX-COX-2 pathway affects tumor growth in vivo using a xenograft model based on MDA-MB-231 cells, a highly aggressive breast cancer cell line naturally overexpressing ACSL4. The first novel finding is that stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system of MCF-7 cells resulted in development of growing tumors when injected into nude mice. Tumor xenograft development measured in animals that received doxycycline resulted in tumor growth inhibition. The tumors presented marked nuclear polymorphism, high mitotic index and low expression of estrogen and progesterone receptor. These results demonstrate the transformational capacity of ACSL4 overexpression. We examined the effect of a combination of inhibitors of ACSL4, LOX-5 and COX-2 on MDA-MB-231 tumor xenografts. This treatment markedly reduced tumor growth in doses of these inhibitors that were otherwise ineffective when used alone, indicating a synergistic effect of the compounds. Our results suggest that these enzymes interact functionally and form an integrated system that operates in a concerted manner to regulate tumor growth and consequently may be potential therapeutic targets for the control of proliferation as well as metastatic potential of cancer cells.     

17β-Estradiol inhibits prostaglandin E2-induced COX-2 expressions and cell migration by suppressing Akt and ERK1/2 signaling pathways in human LoVo colon cancer cells

Epidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2)-induced cellular motility in human colon cancer cells. Upregulation of cyclooxygenase-2 (COX-2) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), or QNZ (NFκB inhibitor), we found that PGE2 treatment increases COX-2 via Akt and ERK1/2 pathways, thus promoting cellular motility in human LoVo cancer cells. We further observed that 17β-estradiol treatment inhibits PGE2-induced COX-2 expression and cellular motility via suppressing activation of Akt and ERK1/2 in human LoVo cancer cells. Collectively, these results suggest that 17β-estradiol treatment dramatically inhibits PGE2-induced progression of human LoVo colon cancer cells.     

Potential misidentification of cyclooxygenase-2 by western blot analysis and prevention through the inclusion of appropriate controls

Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.     

Cyclooxygenase 2 inhibition promotes IFN-gamma-dependent enhancement of antitumor responses

In previous studies, we demonstrated an immune suppressive network in non-small cell lung cancer that is due to overexpression of tumor cyclooxygenase 2 (COX-2). In this study, we assessed the vaccination response to tumor challenge following either pharmacological or genetic inhibition of COX-2 in a murine lung cancer model. Treatment of naive mice with the COX-2 inhibitor, SC-58236, skewed splenocytes toward a type 1 cytokine response, inducing IFN-gamma, IL-12, and IFN-gamma-inducible protein 10, whereas the type 2 cytokines IL-4, IL-5, and IL-10 remained unaltered. Fifty percent of mice receiving SC-58236 and an irradiated tumor cell vaccine completely rejected tumors upon challenge. Those mice that did form tumors following challenge demonstrated a reduced tumor growth. In contrast, all mice either vaccinated with irradiated tumor cells alone or receiving SC-58236 alone showed progressive tumor growth. Studies performed in CD4 and CD8 knockout mice revealed a requirement for the CD4 T lymphocyte subset for the complete rejection of tumors. To determine the role of host COX-2 expression on the vaccination responses, studies were performed in COX-2 gene knockout mice. Compared with control littermates, COX-2(-/-) mice showed a significant tumor growth reduction, whereas heterozygous COX-2(-/+) mice had an intermediate tumor growth reduction following vaccination. In vivo depletion of IFN-gamma abrogated the COX-2 inhibitor-mediated enhancement of the vaccination effect. These findings provide a strong rationale for additional evaluation of the capacity of COX-2 inhibitors to enhance vaccination responses against cancer.     

A novel nitro-oxy substituted analogue of rofecoxib reduces human colon cancer cell growth

Rofecoxib is a specific COX-2 inhibitor able to exert antiproliferative activity against colorectal cancer cells. It was withdrawn from the market after the demonstration of an increased risk of cardiovascular complications after prolonged use. Nevertheless, it remains an interesting compound for laboratory research as an experimental COX-2 inhibitor. In this study, the antiproliferative activity of a novel dinitro-oxy-substituted analogue of rofecoxib (NO-rofe), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with the action of the parent drug. Due to the fact that COX-2 inhibition is the main characteristic of coxibs, we performed all experiments in COX-2-overexpressing (HT-29) and COX-2-negative (SW-480) human colon cancer cells, to elucidate whether the observed effects were dependent on COX-2 inhibition. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signaling pathway. NO-rofe exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the rofecoxib analogue retained similar potencies with respect to COX-2 inhibition but was much more active than rofecoxib in inhibiting the growth of human colon cancer cells in vitro. In addition, this novel compound resulted in the induction of membrane β-catenin/E-cadherin expression, a feature that may significantly contribute to its antiproliferative activity.     

TREM-1 Is Induced in Tumor Associated Macrophages by Cyclo-Oxygenase Pathway in Human Non-Small Cell Lung Cancer

It is increasingly recognized that the tumor microenvironment plays a critical role in the initiation and progression of lung cancer. In particular interaction of cancer cells, macrophages, and inflammatory response in the tumor microenvironment has been shown to facilitate cancer cell invasion and metastasis. The specific molecular pathways in macrophages that immunoedit tumor growth are not well defined. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a member of the super immunoglobulin family expressed on a select group of myeloid cells mainly monocyte/macrophages. Recent studies suggest that expression of TREM-1 in tumors may predict cancer aggressiveness and disease outcomes in liver and lung cancer however the mechanism of TREM-1 expression in the setting of cancer is not defined. In this study we demonstrate that tumor tissue from patients with non-small cell lung cancer show an increased expression of TREM-1 and PGE₂. Immunohistochemistry and immunofluorescence confirmed that the expression of TREM-1 was selectively seen in CD68 positive macrophages. By employing an in vitro model we confirmed that expression of TREM-1 is increased in macrophages that are co-cultured with human lung cancer cells. Studies with COX-2 inhibitors and siCOX-2 showed that expression of TREM-1 in macrophages in tumor microenvironment is dependent on COX-2 signaling. These studies for the first time define a link between tumor COX-2 induction, PGE₂ production and expression of TREM-1 in macrophages in tumor microenvironment and suggest that TREM-1 might be a novel target for tumor immunomodulation.     

Cyclooxygenase-2 up-regulates CCR7 via EP2/EP4 receptor signaling pathways to enhance lymphatic invasion of breast cancer cells

Recent studies demonstrate that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis. However, the mechanism by which COX-2 increases the invasion of cancer cells to lymph node is unclear. CCR7 is a chemokine receptor that plays important roles in the mediation of migration of leukocytes and dendritic cells toward lymphatic endothelial cells (LECs) that express receptor ligand CCL21. We found that treatment of prostaglandin E(2) or ectopic expression of COX-2 in MCF-7 cells up-regulated CCR7 expression. On the contrary, knockdown of COX-2 by small hairpin RNA reduced CCR7 in COX-2-overexpressing MDA-MB-231 cells. Interaction of CCR7 and CCL21 was important for the migration of breast cancer cells toward LECs because antibodies against these two molecules inhibited the migration. We also found that COX-2 increased CCR7 expression via the EP2 and EP4 receptor in breast cancer cells. EP2 and EP4 agonists stimulated CCR7 in MCF-7 cells, whereas antagonists or small hairpin RNA of EP2 and EP4 attenuated CCR7 in MDA-MB-231 cells. Protein kinase A and AKT kinase were involved in COX-2-induced CCR7. Pathological analysis demonstrated that COX-2 overexpression was associated with CCR7, EP2, and EP4 expressions in breast tumor tissues. In addition, CCR7 expression in COX-2-overexpressing tumors was significantly correlated with lymph node metastasis. Collectively, we suggest that CCR7 is a down-stream target for COX-2 to enhance the migration of breast cancer cells toward LECs and to promote lymphatic invasion.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

PPARgamma physiology and pathology in gastrointestinal epithelial cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed at very high levels in the gastrointestinal epithelium. Many of the functions of PPARgamma in gastrointestinal epithelial cells have been elucidated in recent years, and a pattern is emerging which suggests that this receptor plays an important role in gastrointestinal physiology. There is also strong evidence that PPARgamma is a colon cancer suppressor in pre-clinical rodent models of sporadic colon cancer, and there is considerable interest in exploitation of PPARgamma agonists as prophylactic or chemopreventive agents in colon cancer. Studies in mice and in human colon cancer cell lines suggest several mechanisms that might account for the tumor suppressive effects of PPARgamma agonists, although it is not in all cases clear whether these effects are altogether mediated by PPARgamma. Conversely, several reports suggest that PPARgamma agonists may promote colon cancer under certain circumstances. This possibility warrants considerable attention since several million individuals with type II diabetes are currently taking PPARgamma agonists. This review will focus on recent data related to four critical questions: what is the physiological function of PPARgamma in gastrointestinal epithelial cells; how does PPARgamma suppress colon carcinogenesis; is PPARgamma a tumor promoter; and what is the future of PPARgamma in colon cancer prevention?     

New perspectives on APC control of cell fate and proliferation in colorectal cancer

Aberrant Wnt/β-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Considerable evidence for this model has come from mouse models of Apc truncation where nuclear β-catenin is detectable soon after loss of Apc. However, examination of tumors from familial adenomatous polyposis coli (FAP) patients has failed to confirm the presence of nuclear β-catenin in early lesions following APC loss despite robust staining in later lesions. This observation presents the possibility that colon adenomas arise through a β-catenin-independent function of APC. Additionally, there is a well established role for inflammation and specifically COX-2 and prostaglandin E2 in the progression of colorectal cancer. Here we review the current literature regarding the functions of APC in regulating WNT/β-catenin signaling as well as its control of intestinal cell fate and differentiation. Further, we provide a brief commentary on our current understanding of the role that inflammation plays in colorectal tumorigenesis and how it fits in with APC dysfunction. Though there are currently contrasting models to explain colon tumorigenesis, our goal is to begin to reconcile data from multiple different model systems and provide a functional view into the initiation and progression of colon cancer.     

Potential role of group X secretory phospholipase A(2) in cyclooxygenase-2-dependent PGE(2) formation during colon tumorigenesis

Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.     

Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E(2), and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.