IGF1R and MAPK15 Emerge as Potential Targets of Pentabromobenzylisothioureas in Lung Neuroendocrine Neoplasms

Pentabromobenzylisothioureas are antitumor agents with diverse properties, including the inhibition of MAPK15, IGF1R and PKD1 kinases. Their dysregulation has been implicated in the pathogenesis of several cancers, including bronchopulmonary neuroendocrine neoplasms (BP-NEN). The present study assesses the antitumor potential of ZKKs, a series of pentabromobenzylisothioureas, on the growth of the lung carcinoid H727 cell line. It also evaluates the expression of MAPK15, IGF1R and PKD1 kinases in different BP-NENs. The viability of the H727 cell line was assessed by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and its proliferation by BrdU (5-bromo-2′-deoxyuridine) assay. Tissue kinase expression was measured using TaqMan-based RT-PCR and immunohistochemistry. ZKKs (10−4 to 10−5 M) strongly inhibited H727 cell viability and proliferation and their antineoplastic effects correlated with their concentrations (p < 0.001). IGF1R and MAPK15 were expressed at high levels in all subtypes of BP-NENs. In addition, the SCLC (small cell lung carcinoma) patients demonstrated higher mRNA levels of IGF1R (p = 0.010) and MAPK15 (p = 0.040) than the other BP-NEN groups. BP-NENs were characterized by low PKD1 expression, and lung neuroendocrine cancers demonstrated lower PKD1 mRNA levels than carcinoids (p = 0.003). ZKKs may suppress BP-NEN growth by inhibiting protein kinase activity. Our results suggest also a possible link between high IGF1R and MAPK15 expression and the aggressive phenotype of BP-NEN tumors.


Kinase Inhibition Profile of ZKKs
The structurally related ZKKs exhibited multi-kinase inhibitor activity, targeting several kinases known to play key roles in the metabolism of normal and malignant cells, i.e., IGF-1R, ERK8 and PKD1 [9] (Figure 2).

Kinase Inhibition Profile of ZKKs
The structurally related ZKKs exhibited multi-kinase inhibitor activity, targeting several kinases known to play key roles in the metabolism of normal and malignant cells, i.e., IGF-1R, ERK8 and PKD1 [9] (Figure 2).  The selectivity profile of ZKK3 (10 −5 M) determined against a large kinase panel [9]. Residual activity of kinases is shown as percentage of the control without inhibitor. X ± SD.

MRNA Levels of IGF1R, MAPK15 and PKD1 in BP-NEN Patients' Tumors
Forty-five patients with good quality total RNA were chosen for analysis. In the whole BP-NEN group, the highest mRNA expression was demonstrated by IGF1R (RQ 6.66 (3.92-11.92)), which was  [9]. Residual activity of kinases is shown as percentage of the control without inhibitor. X ± SD.

Protein Levels of IGF1R, MAPK15 and PKD1 in BP-NEN Tumors
In BP-NEN patients, the most frequent immunoreactivity was observed for IGF1R kinase, being present in 53 (90%) samples ( Figure 4A). Positive immunoreactivity for MAPK15 was detected in 49
No statistically significant differences in IGF1R, MAPK15 and PKD1 protein levels were observed between different BP-NEN entities. However, IGF1R protein tended to be particularly abundant in SCLC, as it was found to be present in 23 (96%) specimens, of which 11 (46%) showed the most intensive staining (IRS 3). IGF1R immunoreactivity was also detected in the TC group, in which 10 (62.5%) samples were characterized by IRS 2. Most MAPK15 protein expression in BP-NENs had an intensity of IRS 1, which was observed in nine (56%) TC, four (66.7%) AC, nine (37.5%) SCLC and six (46%) LCNEC. However, MAPK15 protein expression tended to be greater in the SCLC sections: only in this group, 13 samples (56%) were found to demonstrate IRS2 and IRS3.
No significant associations were found between mRNA and protein levels for the studied kinases. No statistically significant differences in IGF1R, MAPK15 and PKD1 protein levels were observed between different BP-NEN entities. However, IGF1R protein tended to be particularly abundant in SCLC, as it was found to be present in 23 (96%) specimens, of which 11 (46%) showed the most intensive staining (IRS 3). IGF1R immunoreactivity was also detected in the TC group, in which 10 (62.5%) samples were characterized by IRS 2. Most MAPK15 protein expression in BP-NENs had an intensity of IRS 1, which was observed in nine (56%) TC, four (66.7%) AC, nine (37.5%) SCLC and six (46%) LCNEC. However, MAPK15 protein expression tended to be greater in the SCLC sections: only in this group, 13 samples (56%) were found to demonstrate IRS2 and IRS3.
No significant associations were found between mRNA and protein levels for the studied kinases.

Survival Analyses
A total of 23 deaths occurred within the BP-NEN group; median overall survival (OS, (IQR)) was 1.4 years (0.1-11.00). The median OS within studied subgroups was 2.4 years (1.6-2.7) for TC, 4.2 years (3.0-9.7) for AC, 2.1 years (0.9-2.6) for LCNEC and 0.7 years (0.3-1.2) for SCLC ( Table 1). The differences in OS between BP-NEN entities were significant (p = 0.0002). No patients in the TC or AC groups died. Only borderline significant differences in OS were observed between LCNEC and SCLC groups (p = 0.054). Further statistical analysis of survival was performed only in the lung NEC subgroup and revealed that patients with positive PKD1 protein expression demonstrated shorter OS than those with no PKD1 expression (HR 15.09 (1.68-135.94), p = 0.016). In turn, the lung NET subgroup was further examined on the basis of recurrence data. Tumor relapses were reported in the TC group (one case) and in the AC group (two cases); however, due to the small number of events, they could not be analyzed statistically.

Discussion
The present study shows that ZKK1, ZKK2 and ZKK3 may be effective against BP-NEN in vitro. This is the first study to assess mRNA and protein expression of IGF1R, MAPK15 and PKD1 kinases in all four entities of BP-NENs. Our findings indicate that IGF1R and MAPK15 were abundantly expressed in BP-NEN tissues and that they were present at higher levels in SCLC than in other entities.
ZKKs are compounds with diversified properties, including inhibition of IGF1R, MAPK15 and PKD1 kinases [9]. ZKKs have demonstrated antineoplastic potential against various malignancies; for example, ZKK1-3 (1 × 10 −5 − 5 × 10 −5 M) have been found to inhibit the growth of human leukemia, glioblastoma and prostate cancer cell lines in a time-and concentration-dependent manner [8][9][10]. Structural modifications of ZKK1 influence its efficacy, with N-substitution with short alkyl groups increasing cytotoxic activity [10], as demonstrated by ZKK2 and ZKK3 [8][9][10]. Similarly, in the present study, the most potent cell growth suppression was induced by ZKK3, which completely arrested H727 carcinoid growth at its highest concentration (10 −4 M). The H727 cell line has been previously reported to express IGF1R and PKD1 kinases [24,39], which suggests that ZKKs may suppress growth by inhibiting these kinases. In order to explore this further in a broader context, the study assessed the expression of kinases targeted by ZKKs in different BP-NEN tissues.
Several immunohistochemical studies revealed the presence of IGF1R protein in SCLC tissues [21,[41][42][43]. These papers indicated similar percentages of positive immunostaining for IGF1R in SCLC specimens, ranging from 71 to 81% of SCLC samples, with intensity varying from weak to moderate. In the present study, positive immunoreactivity for IGF1R was observed in 96% SCLC tissues, most of which demonstrated strong staining intensity. Similarly, no previous studies have found IGF1R expression to have any prognostic value in SCLC patients [42,43], except in one cohort with extensive SCLC, in which IGF1R expression was found to be associated with longer OS [42]. Similarly to our present findings, IGF1R expression has previously been demonstrated in other BP-NENs, with no difference in IGF1R level observed between SCLC and LCNEC [21] and between TC and AC tissues [22][23][24]44]. However, our work is the first to offer a complete comparative analysis of IGF1R expression between all four BP-NEN entities and suggest a link between higher IGF1R levels and the aggressive phenotype of tumors. This is an important finding, as in vitro and in vivo studies have found IGF1R overexpression to promote the growth of SCLC [41,45] by interacting with the apoptosis-related Bcl-2 protein family members and AKT pathway [41].
The role of MAPK15 in carcinogenesis, including lung neoplasms, remains relatively understudied and ambiguous. RT-PCR and Western blot analyses have revealed relatively high MAPK15 expression in a few non-small cell lung cancer (NSCLC) lines [46]; however, MAPK15 levels were found to be clearly detectable in lung biopsies of normal tissues but markedly lower in lung carcinomas, including one case of SCLC [36]. MAPK15 expression was also detected by immunohistochemical staining in an atypical carcinoid [47]. Our present findings indicate that MAPK15 is moderately expressed in all types of BP-NEN, with higher levels in SCLC tissues; however, as it is the activation of the signaling pathway that is a fundamental requirement in transformation, not the overexpression itself, the clinical relevance of our findings remains unclear.
Similarly, the impact of PKD1 in lung neoplasms is unclear due to the paucity of studies and often contradictory results. PKD1 has been shown to be down- [48] or upregulated [38] in NSCLC. It has been proposed that cooperation of PKD1 with E-cadherin or the mTORC1/S6K1 (mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase 1) pathway has an antitumor effect in NSCLC [48,49], while PKD1 activation through a PKC-dependent pathway has been shown to mediate pro-tumor responses in SCLC cells [50]. Our study found PKD1 mRNA levels to be higher in lung NETs than in lung NECs. Similarly, the PKD1 mRNA expression has been found to be characteristic for carcinoids and included in the NET-specific transcript set, which could be useful in the diagnostic and monitoring procedures [39]. However, in our tumor samples, PKD1 protein was not expressed, or expressed at low levels. Intriguingly, positive PKD1 protein expression was associated with worsened OS in lung NEC patients.
Interestingly, the statistical analysis did not reveal any significant relationships between the mRNA and protein levels of particular kinases. However, a correlation between increased mRNA and protein expression in SCLC was observed for IGF1R and MAPK15. Importantly, such disassociations between the levels of mRNA and the protein product are commonly observed and connected with post-transcriptional, translational and protein degradation regulation [51,52].
Discussing the limitations of our pilot study, it is important to refer to the relatively small size of the study cohort. This was necessitated by the very low incidence of BP-NENs, especially lung NETs, and the fact that the study was restricted to a single center. In addition, it is difficult to obtain adequate primary SCLC samples for expression studies, as surgery is not a standard therapeutic option for this entity. However, our results of survival analysis are in line with global survival data in BP-NEN patients, indicating that despite its limited size, the cohort is representative for the general population. Moreover, the use of only one cell line can be perceived as a limitation of our study. However, it is important to emphasize that there are only two commercially available cell lines representing lung typical carcinoid, from which the H727 line is the most popular. The fact of limited pulmonary carcinoid cell line resources hinders efforts to understand the biology of this rare type of lung tumor and develop new drugs. In this context, we believe that our results are of value to be reported.
Our results may be important in the therapeutic context. Although targeting the IGF axis in different malignancies has yielded disappointing results in phase III trials [20] and research on MAPK15 targeting drugs is at an very early stage [29], these strategies are still regarded as promising alternatives in precision oncology. Our findings in the tumoral tissues, as well as the in vitro experiments with ZKK multi-kinase inhibitors, suggest that inhibition of IGF1R and MAPK15 could also be potentially effective in lung NET treatment.
ZKK1, ZKK2 and ZKK3 were dissolved in 96% ethanol and serum-free culture medium and added to the appropriate wells at final concentrations of 10 −4 − 10 −6 M (the highest concentration of ethanol was 1.5% (vol) in the 10 −4 M wells). Three control groups (corresponding to 10 -4 ,10 -5 or 10 -6 M ZKK groups) with different ethanol concentrations were used in every experiment. For this purpose, serum-free culture medium and 96% ethanol at the same concentrations as the solvent in corresponding ZKK group were added to the control wells. Each experiment was repeated independently at least twice, with seven biological replicates per group.

Inhibition of Protein Kinases by ZKKs
Kinase profiling for the series of structurally similar ZKKs (i.e., ZKK1-3) was performed with the example of ZKK3. The inhibitory activity of 10 −5 M ZKK3 was tested against a panel of 130 selected protein kinases; the compound was screened in duplicate. The analysis was performed in the International Centre for Kinase Profiling (the University of Dundee, Scotland).

Cell Viability Study (Mosmann Method)
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) colorimetric assay was used as an indicator of cell viability/cytotoxicity (EZ4U kit, Cell Proliferation & Cytotoxity Assay, Biomedica; Wien, Austria; Cat. No BI-5000). The MTT analysis on H727 cells was performed as previously reported [53], but with a density of 1.5 × 10 4 cells per well. The optical density (OD) of each sample was measured by a microplate reader at 450 nm. The results of the MMT analysis were presented as percentage of OD in the corresponding control group.

Cell Proliferation Study
H727 cell proliferation was quantified using colorimetric immunoassay based on the measurement of 5-bromo-2 -deoxyuridine (BrdU) incorporation during DNA synthesis (Cell proliferation ELISA, BrdU, Roche; Penzberg, Germany; Cat. No 11 647 229 001). H727 cell proliferation analysis was performed as previously reported [53]; however, a density of 1.5 × 10 4 cells per well was used. The optical density (OD) of each sample was measured by a microplate reader at 450 nm. The results were presented as percentage of OD in the corresponding control group.

Study Cohort
The study group comprised 49 patients (27 males, 22 females) with median age of 65 years (60.00-70.00) ( Table 2). Samples were provided as 60 formalin-fixed, paraffin-embedded tumor blocks (FFPEs) from the Medical University of Lodz, Poland. All tumors were classified according to the WHO 2004 or 2015 classification of lung neoplasms [54,55]. The study was approved by the Institutional Board of the Medical University of Lodz (Number RNN/145/18/KE). All participants had been recently diagnosed with BP-NENs (2008 to 2019): 11 patients were diagnosed with TC, 5 with AC, 22 with SCLC and 11 with LCNEC. As no significant differences were found between the patients with regard to valid clinical parameters, all patients were further divided into two groups with NETs (n = 16) and NECs (n = 33) for statistical analysis.
Individual clinical data of patients are available in Supplementary Materials (Table S1).

Immunohistochemistry for IGF1R, MAPK15 and PKD1 Protein Expression
Immunohistochemical protein expression was studied in FFPE samples using Mouse Monoclonal (7e12) Anti-Polycystin 1/PC1 Antibody (Abcam; Cambridge, UK; Cat. No AB74115), Rabbit Polyclonal MAPK15 Antibody (ElabScience; Houston, TX, USA; Cat. No E-AB-40233) and Rabbit Policlonal Anti-IGF1R Antibody (Sigma-Aldrich; Saint Louis, MI, USA, Cat. No AB-1161). The following positive controls were set up: MAPK15 and PKD1 on renal carcinoma, and IGF1R on breast cancer. All immunohistochemical procedures were performed as previously reported [56]. Cytoplasmic staining was considered for the evaluation of MAPK15 expression, cytoplasmic and nuclear for IGF1R expression and cytoplasmic and membranous for PKD1 expression. Negative MAPK15, IGF1R and PKD1 staining in tumor sections was defined as IRS 0; positive samples were evaluated as IRS 1, 2 or 3 according to staining intensity. Following this, IRS 0 and 1 were combined into one group and IRS 2 and 3 into a second for statistical analysis.
The stained sections were examined by a BX43 Light Microscope (OLYMPUS Europa SE; CO, Hamburg, Germany). Images were recorded using an UltraFast Scanner (Philips IntelliSite Solution, Best, The Netherlands) with DigiPath™ Professional Production Software (Xerox, Norwalk, CT, USA).

Statistical Analysis
The normality of distribution of continuous variables was determined using the Shapiro-Wilk test. Normally distributed variables were compared using the Student's t-test or one-way analysis of variance (ANOVA) and non-normally distributed variables with the Mann-Whitney U-test (or ANOVA Kruskall-Wallis). Categorical variables were tested using the χ2, two-tailed Fisher's or Yates exact test. Bonferroni's correction was used for multiple comparison. The degree of correlation was tested using Spearman's rank or Pearson coefficient. For the outcome analyses, overall survival (OS) was defined as the time period from diagnosis to last follow-up, with censoring of live patients at the last follow-up. OS data are presented as Kaplan-Meier survival curves. The OS values within subgroups were compared with the log-rank test. P-values < 0.05 were considered statistically significant. Statistical analysis was carried out using the Statistica 13.1 PL package (StatSoft, Tulsa, OK, USA).

Conclusions
Our findings indicate that ZKKs effectively inhibit the growth of bronchial neuroendocrine neoplasms in vitro and that BP-NEN are characterized by abundant expression of the ZKK targets IGF1R and MAPK15. These preliminary but complementing results legitimize further functional research in the field of MAPK15 and IGF1R as potential targets in BP-NEN.