Background
Lung cancer is one of the leading causes of cancer-related death worldwide [
1,
2]. Epidemiological studies report that lung chronic inflammation initiate/promote the development of lung cancer, possibly in conjunction with tobacco use and/or other environmental pollutants (i.e. asbestos, silica, diesel exhaust). Epithelial cells, alveolar macrophages (MФ) and resident dendritic cells (DCs) are the first line of defense for the respiratory tract. Their prolonged contact with insulting exogenous molecules can initiate and sustain inflammatory responses which signature could be IL-1β dependent [
3,
4], leading to chronic inflammation [
3]. In support, elevated serum levels of C-reactive protein (CRP) and high erythrocyte sedimentation rate (ESR) are both associated to lifestyle (i.e. smoking, air pollutant exposure) and are related to increased risk of lung cancer [
5]. Concomitantly, high levels of the pro-inflammatory cytokines, such as IL-1β and IL-18, are detected in the plasma and tissue of lung cancer patients [
6], identified as bad prognostic biomarkers for cancer patients [
7]. IL-1-like cytokines (i.e. IL-1α, IL-1β, IL-18 and IL-33) are identified as ‘alarmins’. Their expression is tightly regulated by multiprotein complexes referred to as ‘inflammasomes’, which activation promotes caspase-1 cleavage into its active form with the ensuing activation of IL-1β and IL-18 [
8]. Alternatively, non-canonical inflammasome engages caspase-11 (also known as caspase-4 in humans) which can induce the release of alarmins such as IL-1α, IL-1β, IL-18 and HMGB1 [
8]. Human caspase-4, as well as the analogue murine caspase-11, was described as a pro-inflammatory caspase that can serve as host defense via the induction of pyroptosis to eliminate intracellular pathogens, and via the release of pro-inflammatory IL-1-like cytokine (i.e. IL-1α and IL-1β, IL-18) in a canonical inflammasome pathway. Nevertheless, in this latter case it was demonstrated that caspase-11 unlikely processes IL-1β and IL-18 in a direct manner [
9], rather, it can promote the downstream caspase-1 activation via NLRP3 [
9]. On the other hand, IL-1α release can be directly related to caspase-11 [
10].
In our previous murine study, we reported that tumor-associated macrophages (TAMs) populated lung tumor lesions exerting a pro-tumor activity in a caspase-11/caspase-1-dependent manner, implying that the activation of the inflammasome in TAMs was pro-tumorigenic [
11]. Moreover, we found that NSCLC patients had higher circulating levels of caspase-4 than healthy subjects [
12]. In this study we demonstrated that caspase-4 was highly present in the tumor mass compared to non-cancerous tissues of NSCLC patients and was responsible for cell proliferation, suggesting it as a novel oncoprotein that collaborates with c-MyC and K-Ras to promote lung cancer, affecting patients’ survival rate.
Materials and methods
Human samples
Samples in this study were obtained by patients diagnosed of operable NSCLC (stage IA-IB, n = 79; Stage IIA-IIB, n = 34; Stage IIIA-IV, n = 12), and underwent surgical resection at Ospedale dei Colli, AORN, Monaldi, Naples, Italy, during the period 2014–2017. Clinical data were obtained from questionnaires and histology reports from the Pathological Anatomy Unit of the hospital. The project was approved by the institutional review board and by the Ethical Committee (approval number for lung cancer patients 1254/2014). Samples from lung cancer patients were collected after oral and written information provided by the MDs, and after the signature of a consent form before entering the project. Samples were collected and used within 24 h. Lung cancer patients were 60 ± 10 (mean ± S.E.M.) years of age. Biochemical analyses on PD-L1 and genetic mutations (i.e. EGFR mutations, ALK, ROS1 and MET genetic alterations) were performed by double blinded operator at Ospedale dei Colli, AORN, Monaldi, Naples, Italy. Survival data was analysed according to the period 2014–2017 and was related to the values of tissue caspase-4 after surgical resection. Human samples were collected and processed within 24 h surgical resection.
Mice
Female specific pathogen-free C57BL/6 mice, B6N.129S2-Casp1 < tm1Flv>/J, CASP1/11 knockout (ko), C3H/HeJ (6–8 weeks of age) (Jackson Laboratories, USA, and Charles River Laboratories, Lecco, Italy) were fed a standard chow diet and housed under specific pathogen-free conditions at the University of Salerno, Department of Pharmacy. CASP11 ko were kindly provided by Dr. Vishva Dixit from Genentech USA. Samples from transgenic mice K-RasLA1 or K-RasLA1/p53R172HΔ were kindly provided by Dr. Quaglino, University of Turin, Italy. All animal experiments were performed under protocols that followed the Italian and European Community Council for Animal Care (2010/63/EU). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Salerno and by National Institutes of Health with the approval number 13786/2014.
Experimental protocol
Mouse model of lung carcinogenesis
Mice were intratracheally (i.t.) instilled with N-methyl-N-nitroso-urea (NMU) at the dose of 50 μg/mouse at week 1 (day 0), week 8 (day 56), week 12 (day 84), and the dose of 10 μg/mouse was instilled at week 1 (day 7), week 2 (day 14), week 9 (day 63), week 10 (day 70), week 13 (day 91) week 14 (day 98), according to Fig.
4a. Lungs were isolated and digested with 1 U/mL collagenase (Sigma Aldrich, Milan, Italy). Cell suspensions were passed through 70 μm cell strainers, and red blood cells were lysed. Cell suspensions were used for flow cytometric analyses of different cell subtypes. Broncho-alveolar lavage fluid (BAL) was collected using 0.5 ml of PBS containing 0.5 mM EDTA and cell counts performed. In addition, lungs were homogenized and cytokines measured.
Bone-marrow (BM) transplantation
Bone-marrow (BM) transplant experiments were performed using wild type (wt, C57Bl/6 mice) and Caspase-11 ko mice. BM-derived cells were isolated from euthanized donor mice. Recipient 6–8 weeks old mice were irradiated with one dose of 10Grad to deplete endogenous BM stem cells and most of the BM-derived cells, before the transplantation of 1 × 106 donor BM cells, injected into the tail vein of recipient irradiated mice.
Animals were divided in four groups:
1.
wt into wt: donor wt cells into recipient wt mice;
2.
ko into ko: donor Caspase-11 ko cells into recipient Caspase-11 ko mice;
3.
wt into ko: donor wt cells into recipient Caspase-11 ko mice;
4.
ko into wt: donor Caspase-11 ko cells into recipient wt mice.
The degree of chimerism was assessed by FACS analysis of CD45.1+ blood leucocytes 7–8 weeks after BM transplant. NMU or vehicle were instilled starting at 8 weeks post BM transplant and chimera mice were sacrificed 28 days after the first NMU exposure.
Flow Cytometry analysis
Cell suspensions obtained by collagenase digested lungs were analysed to evaluate the infiltration and the nature of immune cells recruited to the lung of mice. Cell suspensions were labelled with specific antibodies (CD11b, Gr-1, CD4, CD25, FoxP3).
Western blotting analysis
Lung homogenates were used to examine the expression of caspase-4, in humans, (ImmunePharma srl, Italy) or caspase-11, in mice (Santa Cruz Technologies, CA, USA), kRas (AbCam, Cambridge, UK) by means of SDS- or Native-PAGE. Data were analysed by means of ImageJ (NIH, USA).
ELISAs
The presence of tissue caspase-4 was detected by an ELISA kit patented by ImmunePharma s.r.l. (RM2014A000080 and PCT/IB2015/051262) (Department of Pharmacy, University of Salerno, Italy). Custom antibodies were projected by ImmunePharma s.r.l., and they are not currently commercially available. The diagnostic performance of the custom antibodies has been previously described [
12]. Tissue caspase-4 expression was compared to caspase-5 by means of ELISA. IL-1α and IL-1β were measured in BAL or lung homogenates as specified in the text, using commercially available ELISA kits (eBioscience, CA, USA). In the first case cytokine levels were expressed as pg/ml in BAL samples, whereas in lung homogenates as pg/mg protein.
Immunohistochemistry
Human samples of lung tumor were embedded in paraffin to perform tissue microarray (TMA) Patient’s characteristics are reported in Table
1. NSCLC patients were considered as Caspase-4 positive (+) (Table
1) according to a histological score that was calculated by a blinded and certified pathologist at the National Cancer Institute “Fondazione G. Pascale” (Naples, Italy). In particular, positive score was defined as positive area to caspase-4 detection that resulted ≥25% compared to negative area (≤25%). Human samples analysed by TMA were different from those for whom survival rate is described. A custom antibody against caspase-4, provided by ImmunePharma srl, Italy, was used to perform immunohistochemistry analyses. The diammino-benzidinic acid (DAB) system was used to detect complexes. Mouse IgG was used as an isotype control (ImmunePharma srl, Italy).
Table 1
Characteristics of NSCLC patients and quantification of Caspase-4 positive (+) vs Caspase-4 negative (−) tissues according to the histological score
Age |
≥ 60 yrs | 67 | 53 (79.1%) | 14 (20.9%) |
≤ 60 yrs | 22 | 15 (68.2%) | 7 (31.8%) |
Gender |
Male | 55 | 40 (72.7%) | 15 (27.3%) |
Female | 34 | 25 (73.5%) | 9 (26.4%) |
Stage I | 39 | 31 (79.5%) | 8 (20.5%) |
Stage II | 25 | 16 (64%) | 9 (36%) |
Stage III | 25 | 22 (88%) | 3 (12%) |
Hystotype |
Adenocarcinoma | 54 | 39 (72.2%) | 15 (27.8%) |
Squamous | 32 | 23 (71.9%) | 9 (28.1%) |
Other | 3 | 2 (66.6%) | 1 (33.4%) |
Mice left lung lobes were fixed in OCT medium (Pella Inc., Milan, Italy) and 7 μm cryosections were cut. H&E staining was performed and used to measure the tumour burden. Tumor lesions were analysed by means of Image J (NIH, USA) and expressed as Tumor lesions = ratio tumor area/total lung area, as already reported [
13]. Lung tumor area and the hyperplastic cells were counted by using serial lung cryosections in a blinded fashion.
Reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction
Total RNA was isolated from lung tissue samples by using the RNeasy Mini extraction kit according to the manufacturer’s instructions (Qiagen, United Kingdom). Reverse Transcription was performed by using first-strand cDNA synthesis kit (Qiagen, United Kingdom) followed by PCR, as already reported [
14]. Thermal cycling conditions for caspase-4 were 5 min at 95 °C, followed by 45 cycles of 45 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C.
Thermal cycling conditions for c-MyC were 5 min at 95 °C, followed by 45 cycles of 45 s at 94 °C, 30 s at 66 °C, 30 s at 72 °C.
Primer pairs were as follow:
-Caspase 4 (NM_001225.3): Forward 5′-TTTCTGCTCTTCAACGCCAC-3′; Reverse 5′-AGTCGTTCTATGGTGGGCAT-3′;
-c-MyC (REF NM_002467): Forward 5′-AAAGGCCCCCAAGGTAGTTA-3′; Reverse 5′-GCACAAGAGTTCCGTAGCTG-3′.
-β-actin: Forward 5′-AGAGCTACGAGCTGCCTGAC-3′; Reverse 5′-AGCACTGTGTTGGCGTACAG-3′.
RT-PCR for k-Ras was performed according to the MGBE probe following manufacturer’s instructions (PrimeTime Gene expression Mastermix kit, IDT, USA) were 3 min at 95 °C, followed by 50 cycles of 30 s at 95 °C, 30 s at 54 °C, 30 s at 80 °C. Primers were as follows:
k-RASG12C: Forward 5′-AATATAAACTTGTGGTAGTTGGAGCCT-3′.
k-RASG12D: Forward 5′-AAACTTGTGGTAGTTGGAGCGGA-3′.
k-RASG12V: Forward 5′-AAACTTGTGGTAGTTGGAGCAGT-3′.
k-RAS: Reverse 5′-CATATTCGTCCACAAAATGATTCTG-3′.
Probe: 5′−/56-FAM/CTGTATCGTCAAGGCACT/3MGBEc/− 3′.
Lung cell transfection
A549 cells, adenocarcinomic human alveolar basal epithelial cells, were purchased from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS, L-Glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, Milan Italy) in an atmosphere of 5% CO2 at 37 °C. Cell transfection was performed following manufacturer’s instructions (Transit kit, Mirus Bio Inc., USA). Caspase-4 sequence (NM_001225.3) was encoded into pcDNA3.1 + C-6His and used for cell transfection at the concentration of 50 ng/ml (Genscript Inc., Netherlands). In particular, four different pcDNA plasmids were used according to the caspase-4 mRNA sequence encoded: 1. pcDNA-1 (PC-1): sequence from nucleotide (nt) 74–1205; 2. pcDNA-2 (PC-2): sequence from nt 74–810; 3. pcDNA-3 (PC-3): sequence from nt 348–1205; 4. pcDNA-4 (PC-4): sequence from nt 423–886. Empty vector was used as negative control.
Cell proliferation assay
Transfected and non-transfected A549 cells were previously marked by using carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM; Molecular Probes, Invitrogen) to perform proliferation assay. CFSE flow cytometry data was analyzed by means of ModFit4.0 software (BD Pharmingen). In some experiments, non-transfected A549 cells were treated with human recombinant (ImmunePharma srl., Italy) of the large subunit of caspase-4 (100 ng/ml) and co-cultered with peripheral blood mononuclear cells (PBMCs) obtained by NSCLC patients (ratio 1:5). PBMCs were isolated by means of Ficoll’s protocol as previously reported (Molino et al., 2019).
In another type of experiments, transfected A549 cells were treated with specific pharmacological inhibitors, such as anti-EGFR (10 μg/ml, AbCam, UK, FTI-276 (5 μg/ml, k-Ras inhibitor, Sigma Aldrich, Rome, Italy), SAHA (5 μg/ml, histone deacetilase, HDAC, inhibitor, Sigma Aldrich, Rome, Italy), 5-AZA (5 μg/ml, DNA methylase inhibitor, Sigma Aldrich, Rome, Italy) and rapamycin (1 μg/ml, mTOR inhibitor, Sigma Aldrich, Rome, Italy).
Statistical analysis
Data are reported as median ± percentile range and represented as violin plots. Statistical differences were assessed with TWO-WAY or ONE-WAY Analysis of variance (ANOVA) followed by multiple comparison post-tests as appropriate. Percent survival was estimated by means of Kaplan-Meier method and compared with a non-parametric log-rank test. Percent survival was calculated from the time of surgical resection. The survival rate was calculated for 73 patients which tissue-derived biological samples could be tested by means of the ELISA kit. p values less than 0.05 were considered significant.
Discussion
In this study we found that caspase-4 is correlated to lung carcinogenesis and poor survival rate of NSCLC patients. Herein, caspase-4 could be identified as a novel oncoprotein since 79.3 and 88.2% of adenocarcinoma and squamous NSCLC patients, respectively, stained positive for the protein which pro-tumor activity was reflected in a concerted cooperation with mutated K-Ras and cMyC. Interestingly, a subpopulation of NSCLC patients (20 out of 35 = 57.1%) were triple positive for caspase-4, mutated K-Ras and c-MyC and presented a survival rate of less than 1 year.
Caspase-4 in humans and the analogue murine caspase-11 have been widely described as inflammatory caspases involved in the non-canonical inflammasome pathway in that they are able to sense LPS and lead to the release of IL-1β and IL-18 other than inducing pyroptotic cell death [
8,
28], identified by the release of LDH from cells. One limitation of our study, though, is that the endogenous ligand for caspase-4 in lung cancer is still not identified; however, our data demonstrate that caspase-4/caspase-11 are involved in lung carcinogenesis in humans and mice, respectively. This is the first study, to our knowledge, to show the pro-tumorigenic role of caspase-11 in mice and caspase-4 in humans. Caspase-11 activation is regulated via the TLR4/IFN pathway upon TRIF-induced procaspase-11 processing [
9]. In this regard, we previously demonstrated that the administration of Poly I:C, a TLR3 ligand, that is solely regulated by TRIF and that leads to IFN type I release, reduced tumor burden in a syngenic lung cancer mouse model [
29]. It is noteworthy that Poly I:C-induced reduction of lung tumor in mice was strictly related to the activation of the innate immunity against the tumor. Instead, in this study, we found that caspase-11 is significantly relevant in the structural cell compartment where it is involved in lung carcinogenesis in mice. Indeed, bone marrow transplantation of wild type cells into NMU-treated caspase-11 ko mice, as well as in the case of NMU-treated caspase-11 ko mice, robustly reduced tumor lesions in the lung. Instead, tumor burden of NMU-treated wild type mice that received caspase-11 ko bone marrow cells had higher tumor lesions than NMU-treated caspase-11 ko mice that received wild type bone marrow cells, strengthening what already observed for TAMs which use caspase-11/caspase-1/NLRP3 axis to promote lung tumorigenesis [
11].
In support, a very interesting paper by Cheng et al. [
26], similarly demonstrated the relevance of the non-hematopoietic caspase-11 in a mouse model of lung injury. However, the latter effect was mediated by TLR4. Instead, in our experimental conditions, we found that TLR4 dysfunctional mice (C3H mice) had a similar tumor burden as wild type mice, implying that caspase-11 was not induced by the non-canonical inflammasome pathway. Most likely, because we found that there was a strict correlation between caspase-4 and IL-1α [
4,
12], but not IL-1β, release, we may speculate that caspase-4 activation and IL-1α could be the main orchestrators of lung tumorigenesis in a non-inflammasome-dependent manner in the hematopoietic compartment. It is likely that caspase-4 in the structural cells behaves as an oncoprotein, whereas in the hematopoietic lineage it can allow IL-1α protumorigenic activity. Indeed, the neutralization of IL-1α in NMU-treated mice significantly reduced the levels of tumor areas than control group and, very importantly, NSCLC patients who presented high levels of IL-1α and caspase-4 had lower median survival rate.
Lung tumor-associated caspase-4 was related to tumor cell proliferation, rather than cell death. This effect was correlated to the large subunit of caspase-4. In literature. Caspase-4 and caspase-11 activity are often associated to the release of lactate dehydrogenase, LDH, as a marker of cell death. It is worldwide known that high levels of LDH characterize inflammatory patterns, together with CRP and ERS, which are highly detected in cancer patients. Similarly, in our experimental conditions, we found that all patients positive for tumor-associated caspase-4 had high levels of LDH, which was not a measure of cell death as reported in in vitro assays [
27,
30], but of cancer progression [
31]. To date, LDH is an enzyme that catalyzes the pyruvate to lactate during anaerobic conditions. The metabolomic profile of cancer patients is well-described as altered in that to promote the accumulation of pyruvate which leads to both the anaerobic (LDH-dependent) and tricyclic acid (TCA) pathway according to the tumor cell metabolic needs [
32,
33]. Therefore, tumor-associated as well as circulating caspase-4 [
12] suggest that the inflammatory pattern in tumor cells alters the metabolomic profile to favor cell proliferation rather than cell death, as we recently demonstrated [
31]. In support, Trinidad et al., proved that the activation of the pyruvate kinase 4, which phosphorylates the pyruvate at the last step of glycolysis, is correlated to the metabolic phenotype of K-Ras in favor of tumor cell proliferation [
34]. The oncogenic K-Ras was associated with higher levels of hexokinase 2 (HK2), involved in high-rate metabolism of the glucose in lung cancer-associated TCA according to higher consumption of glutamine [
35].
Similarly, various studies have demonstrated that c-MyC-driven tumors display increased glucose uptake and catabolism to lactate and TCA cycle intermediates [
36]. In this context we found that 85.2% of NSCLC patients who presented K-Ras mutation and 66.7% of patients who overexpressed c-MyC were positive for tissue caspase-4. The median survival rate of these two subgroups of patients was 0.97 (Fig.
3j, black line) and 1 (Fig.
3g, black line) year. However, our study had a limited number of patients at stage III due to the fact that these patients usually undergo therapeutic treatment without surgical resection. In addition, we found that K-ras
LA1 and K-ras
LA1/p53R172HΔ Tg mice had higher lung levels of the cleaved form of caspase-11 (Fig.
4d), associated to higher tumor lesions developed in double Tg mice as reported by Riccardo et al. [
24]. These data together with the in vitro studies (Fig.
5) suggest that caspase-4 underlies K-Ras-mediated cell proliferation. However, it has to be pointed out that Caspase-4+ K-Ras -– (Fig.
3j, blue line; Fig.
3l, 14.8%) or Caspase-4+ cMyC- (Fig.
3g, blue line; Fig.
3i, 33.3%) NSCLC patients still had lower survival rate. In this regard, a recent manuscript demonstrated that caspase-4 can lead to epithelial-mesenchymal transition in lung cancer [
37], further highlighting the importance of our discovery on the role of caspase-4 in lung carcinogenesis in humans.
Another important issue is that we found that among PD-L1 negative patients, 88.1% were positive to caspase-4 (Fig.
3c). Similarly, 89.8% of NSCLC patients who did not present EGFR mutation or ALK, ROS1, MET genetic alterations were positive to caspase-4. Key established predictive biomarkers for target therapy include ALK and ROS1 rearrangements, EGFR mutations, BRAF V600E point mutations, and PD-L1 expression levels [
2]. The National Comprehensive Cancer Network (NCCN) panel recommends testing for these key established biomarkers in patients with NSCLC to decide for the pharmacological treatment, because effective targeted therapies or immunotherapy are available. In particular, tyrosine kinase inhibitors (TKIs) and monoclonal antibodies against PD-1/PD-L1 axis are becoming the first-line therapeutic options for NSCLC patients positive to these targets. However, patients treated with TKIs develop drug resistance after around 12–15 months of treatment. Similarly, patients treated with anti-PD-1/PD-L1 monoclonal antibodies can develop resistance with various mechanisms [
2]. Even worse is the situation of non-mutated NSCLC patients whose sole therapeutic option could be the classical chemotherapy or chemotherapy in addition to anti-PD-L1 antibodies, although the low expression of PD-L1 in tumor tissues. Therefore, here we identified a novel subpopulation of NSCLC patients as caspase-4 positive among which we identified patients as double positive for caspase-4 and K-Ras or c-MyC, and triple positive, identified as caspase-4, K-Ras and c-MyC positive.
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