Cathepsin L activated by mutant p53 and Egr-1 promotes ionizing radiation-induced EMT in human NSCLC
Ionizing radiation (IR) is one of the major clinical therapies of cancer, although it increases the epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC), unexpectedly. The cellular and molecular mechanisms underlying this role are not completely understood.
We used NSCLC cell lines as well as tumor specimens from 78 patients with NSCLC to evaluate p53, Cathepsin L (CTSL) and EMT phenotypic changes. Xenograft models was also utilized to examine the roles of mutant p53 (mut-p53) and CTSL in regulating IR-induced EMT of NSCLC.
Expression of CTSL was markedly increased in human NSCLC tissues with mutant p53 (mut-p53), and p53 mutation positively correlated with metastasis of NSCLC patients. In human non-small cell lung cancer cell line, H1299 cells transfected with various p53 lentivirus vectors, mut-p53 could promote the invasion and motility of cells under IR, mainly through the EMT. This EMT process was induced by elevating intranuclear CTSL which was regulated by mut-p53 depending on Early growth response protein-1 (Egr-1) activation. In the subcutaneous tumor xenograft model, IR promoted the EMT of the cancer cells in the presence of mut-p53, owing to increase expression and nuclear transition of its downstream protein CTSL.
Taken together, these data reveal the role of the mut-p53/Egr-1/CTSL axis in regulating the signaling pathway responsible for IR-induced EMT.
KeywordsNon-small cell lung cancer Ionizing radiation Epithelial-mesenchymal transition p53 mutation Cathepsin L
bovine serum albumin
Early growth response protein-1
fetal bovine serum
non-small cell lung cancer
phosphate buffer saline
squamous cell carcinoma
zinc finger E-box binding homeobox 1
Lung cancer is the most lethal cancer worldwide, and approximately 80% of lung cancers are non-small cell lung cancer (NSCLC) . Radiation therapy is one of the major clinical tools of NSCLC treatment, together with chemotherapy and surgery . Radiotherapy causes DNA damage directly by ionization, thereby destroying cancer cells. However, recent studies indicated that ionizing radiation (IR), paradoxically, promotes invasion and metastasis of NSCLC cells by inducing the epithelial-mesenchymal transition (EMT) [3, 4]. Invasion and metastasis are the main obstacles to successful therapy and are closely linked to the mortality rate of NSCLC. Therefore, the mechanism of IR-induced EMT in NSCLC is needed to be elucidated urgently.
The progress of NSCLC involves multiple genetic abnormalities that lead to EMT of the aggressive bronchial epithelial cells [5, 6]. Among such genetic abnormalities, mut-p53 occurs in about 50% of NSCLC . Apart from the loss of tumor-suppressor functions, mut-p53 may gain new functions independent of wild-type p53 (wt-p53), defined as gain-of-function (GOF), which enable them to contribute to malignant progression, including increased cell invasion and metastasis which are the main traits of EMT [8, 9]. Interestingly, patients whose tumors contain p53 gene present an increase in tumor metastasis when underwent radiation or DNA-damaging reagents . However, a few reports have shown p53 mutation as a delayed effect of radiation, and the correlation between mut-p53 and IR-induced EMT in NSCLC is scarcely known.
Our previous study showed that IR promoted EMT especially in mut-p53 human glioma cells, and the key effector that induces EMT may be Cathepsin L (CTSL) . CTSL, a ubiquitously expressed lysosomal cysteine protease, is primarily involved in terminal degradation of intracellular and endocytosed proteins . Accumulating evidences reveal that CTSL specifically high-expressed in a wide range of human cancers [13, 14, 15, 16]. Simultaneously, our recent study indicated that the expression level of CTSL correlates positively with the degree of tumor malignancy . Moreover, CTSL transported into the nucleus plays an important role in regulating cellular transcription factors, and thus affects the morphology or activity of cancer cells. Notably, the nuclear CTSL activates the transcription of EMT genes and also confers a replicative and metastatic advantage to tumor cells . In fact, we also found that CTSL inhibition could suppress EMT-mediated invasion and metastasis of lung cancer cells . Overall, the role of CTSL in promoting tumor progression and metastatic aggressiveness have raised significant interest in the upstream genes of CTSL intervention strategies. Indeed, one research reported that human CTSL promoter contains two p53 binding motifs and wt-p53 could upregulate CTSL in a static state . However, there was no report which explains the role of mut-p53 in regulating CTSL and the pathway of mut-p53 functions. The fact that mut-p53 facilitates transcription of transformation related genes was shown in several studies . Of note, induction of Early growth response-1 (Egr-1) gene expression was one of the significant changes observed upon overexpression of mut-p53 in human lung cancer cells . The Egr-1 protein is a transcription factor involved in various biological functions including regulation of proliferation, differentiation and apoptosis . It is noteworthy that Egr-1 is a direct transcriptional target of mut-p53 which demonstrated by promoter analysis and chromatin immunoprecipitation assays. Both Egr-1 mRNA and Egr-1 protein levels were upregulated by mut-p53. Interestingly, Egr-1 is not only participates in the GOF effect of mut-p53 but also an IR responsive factor . Furthermore, this stress response gene is involved in CTSL expression regulation under both genetic and metabolic stress conditions in human carcinoma cells . These data suggest that specific regulation of Egr-1 binding on CTSL promoter could be triggered under conditions others than normal culture conditions. In addition, it was described that Egr-1 also showed a different function during EMT in NSCLC . Collectively, these findings show that Egr-1 may be the target protein of mut-p53 in regulating CTSL mediated EMT under IR.
Herein, we consequently proposed a hypothesis that the transcriptional activation of CTSL which was mediated by mut-p53/Egr-1 axis increases the IR-induced EMT. Besides, we have also indicated that expression of CTSL correlates with radioresistant, as well as malignant grades of tumor tissues . Thus, on the basis of our previous findings, it is of scientific significance to study the mechanism of mut-p53 up-regulating CTSL expression and promoting IR-induced EMT of NSCLC cells. Overall, our study defines CTSL as a novel target of mut-p53 under IR and provides new insights for the understanding of mut-p53 GOF activity.
Patients and tumor specimens
A cohort of 78 diagnosed patients with NSCLC was treated at the Affiliated Hospital of Jiangsu University (Zhenjiang, China) between March 2012 and February 2013 to be included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the Ethics Committee of Jiangsu University. Tumor specimens and adjacent tissue samples were immediately snap-frozen in liquid nitrogen after surgical removal. All the specimens were collected for genetic mutation analysis, western blotting and immunohistochemistry assays. The histopathological assessment was carried out separately by two pathologists and ended with a consensus decision. The staging system was carried out according to the 2009 IASLC classification. Brief clinical and pathologic characteristics of the subjects are presented in Additional file 1: Table S1. Besides clinicopathological characteristics, we also collected follow-up information in a subgroup of 60 patients, including treatment, survival status and survival time. Among them, there were 12 patients (20.0%) receiving radiotherapy. The survival time of these patients ranged from 3 to 60 months with the median of 42.9 months.
Cell line and culture conditions
Human NSCLC cell lines (H1299, H1650, VMRC-LCD, H322, and H1838) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies), 100 unit/ml penicillin and 100 units/ml streptomycin. The cells were grown in a humidified incubator with 5% CO2 at 37 °C.
The human and mice cells were irradiated using a 6-MV X-ray linear accelerator (model: PRIMUS, D. E., Siemens, Malvern, PA, USA) at a dose rate of 198 cGy/min. After IR, the cells were placed in the incubator and samples were collected at the indicated time points (0, 6, 12, 24 h).
Genetic mutation analysis
Total RNA was extracted from the fresh-frozen fraction of tumor tissue using Trizol reagent (Invitrogen). RNA samples were then used to synthesize cDNA using a PrimeScript RT reagent Kit (Takara) following the manufacturer’s instructions. Primer sequences (Additional file 2: Table S2) were designed for mutational analysis of exons 5 to 8 of the p53 gene. PCR product sizes were controlled by migration on 2% agarose electrophoresis gel and were purified with DNA gel extraction kit (Axygen). Targeted exon sequences were verified by Sangon Biotech (Shanghai, China).
The ABC detection system (Vectastain Elite ABC Kit, Vector) was used for immunostaining according to the manufacturer’s protocol as described previously . Specific primary antibodies were used as follows: anti-CTSL (Abcam, ab6314), anti-Snail (Santa Cruz, sc-28199), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203) and anti-Vimentin (Abcam, ab92547). The negative control slides, without the primary antibodies, did not exhibit nonspecific staining. In this study, all slides were examined by two pathologists in a blinded fashion. To evaluate the expression level of the proteins, the intensity of staining was scored according to a positive index of tumor cells.
Western blotting was conducted as previously described . The primary antibodies used in this study are as follows: anti-CTSL (Abcam, ab6314), anti-p53 (Abcam, ab1101), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203), anti-Vimentin (Abcam, ab92547), anti-Zeb1 (Abcam, ab203829), anti-Snail (Santa Cruz, sc-28199), anti-Egr-1 (Santa Cruz, sc-189), and loading control anti-β-actin (Abcam, ab8226). Finally, the primary antibodies were probed with rabbit or mouse secondary antibodies labeled with DyLight 800 (KPL) and scanned with the Odyssey Infrared Imaging System (LI-COR).
Construction of point mutation plasmids
The p3XFLAG-Myc-CMV-p53 (Flag-p53) expression vector, which carries wild-type p53, was a generous gift from Professor Wang Guanghui at the Laboratory of Molecular Neuropathology, College of Pharmaceutical Sciences, Soochow University. Flag-p53 (R175H), Flag-p53 (R248Q), and Flag-p53 (R273H) expression vectors, containing p53-R175H, p53-R248Q, and p53-R273H mutants, were generated from Flag-p53 by a MutanBEST Kit (TaKaRa) following the manufacturer’s recommendations. The primers sequences used for mutagenesis were listed in Additional file 3: Table S3. The sequences of the mutation constructs were verified by Sangon Biotech (Shanghai, China). Transfection of these plasmids was performed using Lipofectamine® 2000 Reagent (Invitrogen) according to the manufacturer’s instructions as previously described .
Lentivirus transfection and isolation of stable cell clones
The p53-null H1299 cells were transfected separately with 2 × 107 titration units of lentivirus packaging (Flag lentivirus, Flag-p53 lentivirus, and Flag-p53 (R273H) lentivirus) synthesized by Gene Chem (Shanghai, China). After 2 days of transfection, the medium was replaced with fresh RPMI-1640 containing 10% FBS and 300 μg/mL G418 (Roche). After the dilution culture was limited under the pressure of G418, several independent clones of each transfection group were selected and screened for p53 protein expression by western blotting. Three stable transfected cell clones were selected for further experiments and each designated as Flag, Flag-p53, and Flag-p53 (R273H).
Cells were grown on glass coverslips (Fisher Scientific). After treatment with 8 Gy of IR, cells were fixed with 4% paraformaldehyde. Following the PBS (phosphate buffer saline) wash, cells were permeabilized using 0.2% Triton X-100, incubated in a blocking solution (PBS, 3% BSA (bovine serum albumin)), and further incubated overnight at 4 °C with primary antibodies, including anti-CTSL (Abcam, ab6314), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203) and anti-Vimentin (Abcam, ab92547). Thereafter, Alexa Fluor 488 (Molecular Probes, 1:500) was added as a fluorescent-conjugated secondary antibody. For F-actin staining of the cytoskeleton, cells were stained with FITC-Phalloidin (Thermo Fisher Scientific). DAPI (Sigma Aldrich) was used as a nuclear counterstain. Finally, the coverslips were mounted onto slides with fluorescent mounting medium (Vector Labs) and immediately observed by confocal microscopy (Carl Zeiss, LSM 710).
For the wound-healing assay, the cells were grown in 6-well plates. When a confluent layer was achieved, the cells were scratched using a pipette tip, rinsed to remove debris, and then further incubated with fresh culture medium containing 1% fetal bovine serum (FBS). The initial distance of the wound was recorded at the beginning in order to estimate the recovery of the wounded layer by cell migration, after 24 h the images were captured using an inverted phase-contrast microscope. The wound-healing index, which was determined as a percentage, was quantitatively analyzed using 20 randomly selected distances across the wound from 0 h to 24 h, divided by the distance measured at 0 h. For each experiment, three replicates were performed.
Cell invasion assay
Cell invasion assay was performed using a Boyden chamber containing 24-well transwell plates (Corning) with 8 μm pores on the membrane. All experiments were performed in duplicate and repeated three times. The membrane was coated with 100 μl 1:8 diluted Matrigel (BD Biosciences). After solidifying Matrigel at 37 °C, approximately 1 × 105 cells in 200 μl culture medium supplemented with 1% FBS were seeded into the upper chamber, whereas the lower chamber was filled with the complete medium. Subsequently, the Boyden chamber was incubated at 37 °C with a 5% CO2 atmosphere for 24 h. On the assessment day, cells from the interior of the inserts were removed and the membranes were stained with crystal violet. The cells were counted by photographing the membrane through the microscope.
Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation was carried out by using the EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Briefly, cells were fixed with 1% formaldehyde at room temperature for 10 min to cross-link proteins to DNA. Then, the cross-linked chromatin was sonicated into 200 to 1000 bp fragments and immunoprecipitated using anti-p53 (Abcam, ab1101), anti-Egr-1 (Santa Cruz, sc-189) or IgG control antibodies. The samples were extensively washed after the cross-links were reversed and DNA was purified. The primers used for PCR amplification of specific promoter regions were listed in Additional file 4: Table S4.
CTSL siRNA (Santa Cruz, sc-29938) and Egr-1 siRNA (Additional file 5: Table S5) were obtained from GenePharma (Shanghai, China). For transfection, siRNA was mixed with Lipofectamine® 3000 Reagent (Invitrogen) and then transfected into Flag-p53 (R273H) cells. The entire process was conducted according to the manufacturer’s guidelines. After 6 h, the supernatant was replaced with fresh medium containing 10% FBS and cultured for another 24 h.
All mice experiments were conducted in accordance with the humane treatment of animals under institutional guidelines approved by the Ethical Committee of Soochow University. The mice were housed in individually ventilated cages in the Animal Laboratory of the Medical College of Soochow University. Five-week-old male BALB/c nude mice (Animal experiment Center of Soochow University, Suzhou) were used in the study. Subcutaneous tumor transplantation was conducted using the Flag, Flag-p53, and Flag-p53 (R273H) cells. Cells (n = 1 × 107) were resuspended in 100 μl PBS and implanted into the right flank of nude mice under sterile conditions. After the formation of palpable tumors (tumor volume reached 100 mm3), mice were randomized into six groups (5 mice per group): Flag group, Flag plus IR group, Flag-p53 group, Flag-p53 plus IR group, Flag-p53 (R273H) group, and Flag-p53 (R273H) plus IR group. The size of the tumor and the body weight of each mouse were measured as described previously . The IR treatment was given quartic with a dose of 5 Gy until 20 Gy IR had been delivered. Mice were sacrificed on day 20, and tumor tissues were harvested for western blotting and immunohistochemistry.
All quantitative data are presented as mean ± S.D. from at least three independent experiments per data point. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software). Quantitative variables were analyzed with the Student’s t-test. The chi-square (χ2) test or Fisher exact test was used to compare qualitative variables. The overall survival (OS) of NSCLC patients was estimated using the Kaplan-Meier method. Mantel-Cox’s proportional hazard regression model was established for univariate and multivariate analyses of the combined contribution of p53 and clinicopathological features to the OS of patients. P < 0.05 was considered statistically significant.
p53 mutation promoted metastasis and reduced the survival time of NSCLC patients
The correlation between p53 mutational status and clinicopathologic features in 78 NSCLC patients
WT (n = 35)
Mut (n = 43)
IR promoted cell motility and EMT in the presence of mut-p53 in vitro
EMT process plays a vital role in tumor invasion and metastasis in various solid tumors, particularly, in NSCLC . In order to study whether IR-induced tumor motility in presence of mut-p53 was mediated by EMT, we evaluated the expression level of EMT markers of mut-p53 cells after IR exposure by western blot. The results showed that E-cadherin level decreased significantly in Flag-p53 (R273H) cells (P < 0.05), while N-cadherin level increased (P < 0.01). Similarly, this tendency was more significant under IR. In Flag and Flag-p53 cells, there was no notable change of EMT marker proteins (Fig. 2d). Simultaneously, to further verify the data from western blot, we conducted immunofluorescence staining of cells in order to locate EMT marker and found the same results for E-cadherin and N-cadherin generated by western blotting assay. The EMT-associated proteins, vimentin, had also a clearly elevated expression in Flag-p53 (R273H) cells, and IR further promoted this expression (Fig. 2e). The transcriptional mechanisms for EMT have been well elucidated. The Snail transcription factor enhances expression of the EMT transcription factor zinc finger E-box binding homeobox 1 (Zeb1) and plays a role of master regulator of EMT . PCR assays results showed the mRNA level of other EMT-associated markers, Snail and Zeb1 were both increased in Flag-p53 (R273H) cells, whereas Twist1 showed no change (Fig. 2f). Hence, IR promoted mut-p53 GOF activity of tumor motility might be affected through the process of EMT.
CTSL crucially mediated the IR-induced EMT in the presence of mut-p53
The mut-p53 regulates the transcription of CTSL depended on Egr-1 activation under IR
In order to further determine whether Egr-1 mediated the transcriptional regulation of mut-p53/CTSL, three Egr-1 specific siRNA sequences were used to inhibit the expression of Egr-1 in Flag-p53 (R273H) cells with IR. Western blotting analysis showed that Egr-1-siRNA3 sequence could effectively inhibit the expression of Egr-1 (P < 0.001), and the CTSL expression was restrained obviously after IR (P < 0.01, Fig. 4d). Meanwhile, the mut-p53 could not bind to the promoter of CTSL when Egr-1 gene has successfully interfered (P < 0.01, Fig. 4e). The results above showed that the mut-p53 regulated the transcription of CTSL depended on Egr-1 activation under IR.
CTSL was correlated with p53 mutation and tumor malignancy in NSCLC patients
To better understanding the effect of CTSL on tumor progression, a western blotting analysis was conducted on NSCLC tissues (Additional file 10: Figure S5). As was shown, CTSL was high-expressed in most tumor tissues compared with adjacent tissues (P < 0.001, Fig. 5c). Additionally, the expression of CTSL progressively increased with increasing malignancy grade in squamous cell carcinoma (SCC) (P < 0.01), while not evident in adenocarcinoma (ADC) (Fig. 5d). Furthermore, immunohistochemistry analysis showed CTSL expression was higher in stage III and stage IV of NSCLC (Fig. 5e). Thus, high expression of CTSL was also associated with tumor progression.
CTSL promoted IR-induced EMT in the presence of mut-p53 in vivo
An important finding of this study was that the patients with the p53 mutation were more likely to have tumor metastasis, and the survival rate of IR-treated patients with mut-p53 was significantly decreased. The hot mutant spots of p53 mainly existed on the exon 5–8 and almost were point mutations. Among them, p53 mutated at codon 273 played a vital role in metastasis, with a high metastasis rate of 40%. It was indicated that p53 mutation (particularly in codon 273) could be regarded as a key indicator of tumor metastasis in NSCLC patients.
Tumor metastasis is a complex pathological process that requires the invasion and migration of tumor cells, which are closely related to the EMT-induced movement of the tumor [41, 42, 43]. It was demonstrated here that both, the migration ability and cell motility of mut-p53 cells (codon 273), could be improved by IR. Furthermore, the EMT-associated proteins were also clearly elevated in mut-p53 cells (codon 273), and IR further promotes these expressions. It was suggested that the metastasis of p53-mutated cells increases after IR, through the process of EMT, probably.
CTSL, the main member of the Cathepsins family, not only takes part in regulating invasion and metastasis of tumor [44, 45], but also modulates transcription of EMT related genes . It was controversial that whether CTSL could be regulated by mut-p53 in human glioblastoma cells [11, 18]. In this study, the expressions of CTSL were increased by wt-p53 and remarkably upregulated by IR in mut-p53 lung cancer cells. The up-regulated CTSL in wt-p53 lung cancer cells was mainly mostly distributed in the cytoplasm, while CTSL could be transported from the cytoplasm into nucleus after treated with IR only in the mut-p53 group, playing an important role in affecting the EMT of lung cancer cells. It is well explained that although the elevated expression of CTSL occurred in the wt-p53 tumor, it had a weak influence on p53 GOF activity of tumor motility. In addition, why the CTSL transcribed by mut-p53 could be located in the nucleus, needs further investigation. We suspect that the mechanism may be closely related to the initiation site of p53-regulated CTSL translation . Nevertheless, our previous hypothesis was confirmed, so that IR-activated transcription of CTSL was mut-p53 dependent and affected the EMT of cancer cells.
Most importantly, our data discovered a novel mechanism of the CTSL transcription mediated by the mut-p53 after IR. Firstly, the peaked expression level of Egr-1 in the mut-p53 group was earlier than augment of CTSL expression. On the other hand, Egr-1 could bind to the promoter of CTSL upstream in the mut-p53 group, but not in the wt-p53 group. Similarly, the p53 could bind to Egr-1 only in the mut-p53 group with IR enhancing this interaction. Furthermore, CTSL expression in p53-mutated lung cancer cells was restrained obviously after IR by Egr-1 inhibition. Meanwhile, the mut-p53 could not bind to the promoter of CTSL when Egr-1 gene has successfully interfered. The results showed that mut-p53 regulates the transcription of CTSL depending on Egr-1 activation under IR, but not the wt-p53.
Moreover, our study further verified that CTSL expression level was significantly higher in tumor tissues than in adjacent tissues, and positively related to the tumor grade. Additionally, the expression of nuclear CTSL was dramatically elevated in mut-p53 tissues, as well as EMT-associated proteins, which was in accordance with the result in lung cancer cells. Thus, CTSL can be regarded as a potential target in NSCLC patients.
In addition, the tumor formation of IR-treated groups was significantly reduced in the Flag and Flag-p53 nude mice, whereas the Flag-p53 (R273H) nude mice could not be evidently decreased. Importantly, after exposure to IR, CTSL nuclear location and EMT level of Flag-p53 (R273H) group were increased, consistent with the in vitro studies. It is worth noting that targeting CTSL might be very significant for conquering radiotherapy-induced EMT of lung cancer patients whose p53 gene is mutant.
Our studies indicated that mut-p53 cells become invasive after IR, by a combination of Egr-1-dependent effects, including high levels of CTSL, Snail and Zeb-1 expression that leads to EMT behavior. Thus, CTSL acts not only as an additional diagnostic marker but also as a novel therapeutic target for NCSLC patients with mut-p53.
The authors sincerely thank Dr. Wang Guanghui for providing us with p3XFLAG-Myc-CMV-p53 vector.
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81773768 and 81703532).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
WW, YX and XD conceived and designed the experiments, LW, YZ, YF, YZ, and XS performed experiments and analyzed data, WW and YX wrote the manuscript, CT and ZL revised manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the Ethics Committee of Jiangsu University. The animal studies described were approved by the Ethical Committee of Soochow University.
Consent for publication
All authors approved of the manuscript and consented to its publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 4.Cho JH, Hong WG, Jung YJ, Lee J, Lee E, Hwang SG, et al. Gamma-ionizing radiation-induced activation of the EGFR-p38/ERK-STAT3/CREB-1-EMT pathway promotes the migration/invasion of non-small cell lung cancer cells and is inhibited by podophyllotoxin acetate. Tumour Biol. 2016;37(6):7315–25.PubMedCrossRefGoogle Scholar
- 6.Kerr KM, Lopez-Rios F. Precision medicine in NSCLC and pathology: how does ALK fit in the pathway? Ann Oncol. 2016;27(Suppl 3):iii16–24.Google Scholar
- 18.Katara R, Mir RA, Shukla AA, Tiwari A, Singh N, Chauhan SS. Wild type p53-dependent transcriptional upregulation of cathepsin L expression is mediated by C/EBPα in human glioblastoma cells. Bio Chem. 2010;391(9):1031–40.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.