NFIB controls chemosensitivity in small cell lung cancer by suppressing Notch signaling activity

NFIB controls chemosensitivity in small cell lung cancer by suppressing Notch signaling activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NFIB controls chemosensitivity in small cell lung cancer by suppressing Notch signaling activity Weixin Qin, Ziyan Wang, Shuzhe Deng, Huilei Qiu, Hongxue Meng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3866018/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Small cell lung cancer(SCLC) is a highly aggressive malignant tumor type, characterized by universal acquired therapeutic resistance during rapidly recurrence. However, we have a poor understanding of the mechanisms underlying development of resistance. NFIB is a bona fide oncogene in SCLC with effects on proliferation, invasion, and apoptosis inhibition. Methods: The expression of NFIB and related genes was evaluated in SCLC cells and tissue specimens, by western blot, RT-PCR immunofluorescence and immunohistochemistry. The relationship between genes was verified by ChIP-PCR experiments. CCK8 assays was used for drug resistance experiments. Results: NFIB is highly correlated with NE markers and may be participated in the generating of tumor heterogeneity mediated in part by Notch1. The suppressive effect of NFIB on Notch1 is relieved and led to NE gene inhibiting when SCLC cells lack of NFIB. These cells are slow growing and also relatively chemoresistant. Importantly, Notch blockade in combination with chemotherapy alleviates the formation of intratumoral heterogeneity and enhances chemosensitivity. Conclusions: NFIB can be both tumor-pro-tumorigenic and chemosensitizing in SCLC. NFIB knockdown results in endogenous activation of the Notch pathway. These results uncover the dual character of oncogene as NFIB in SCLC and offer perspectives for efficacious combination therapies that might also hold promise for treating human SCLC. NFIB Notch chemosensitivity heterogeneity SCLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Small cell lung cancer(SCLC) is the most aggressive subtype of lung cancer, which accounts for 13% of all lung cancers, characterized by rapid growth and early metastatic spread 1 . SCLC patients typically present with initial chemosensitivity, followed by the rapid emergence of chemoresistance, the 5-year overall survival (OS) is < 10% 2 . There has been limited improvement in first-line SCLC treatment in the past 30 years and no targeted therapies are currently available 3 . However, mechanisms underlying metastases and rapidly resistance of SCLC cells are not understood, and SCLC remains a recalcitrant cancer. Notch singling plays an important role in cell-fate decisions in a variety of tissues 4 . Activation of Notch family members potently suppresses in SCLC mouse models during lung development intrinsically to NE cells and also can be protumorigenic through produceing non-NE cells that are more chemoresistant 5 – 10 . Activation of the Notch pathway results in a NE to non-NE fate switch occurred in 10–50% of NE cells in SCLC and coincided with the induction of REST, a transcriptional repressor that inhibits NE gene expression 11 . One gene that is regulated by the Notch1 is hairy/ enhancer of split (Hes1), a basic helix-loop-helix transcription factor, which has been described as a negative prognostic factor for overall and progression-free survival in SCLC tumors. In addition, ASCL1, a transcription factor that promotes neuroendocrine transcriptional programs, appears to be a direct downstream target of Notch singling 12 . NFIB belongs to the nuclear factor I (NFI) family of transcription factors which play important roles during lung and brain development and have been associated with developmental abnormalities in humans 13 . The NFI family consists of four transcription factors in humans and most vertebrates: NFIA, NFIB, NFIC, and NFIX, all have a homologous DNA binding domain and function in adenoviral DNA replication and in the regulation of transcription of a large variety of cellular and viral genes 14 . Recent studies have defined NFIB as an oncogene in SCLC 15 , 16 . Through NFIB promotes metastasis through a widespread increase in chromatin accessibility and changes the metastatic profile in a Mouse Model of SCLC, increased expression of the NFIB was identified as a pro-metastatic switch 17 , 18 . While NFIB upregulation may represent critical to metastasis in a subset of human SCLC, the molecular mechanism of NFIB in tumorigenic process and possible associations with sensitivity to chemotherapy, prognosis, and clinical characteristics of SCLC remained elusive. Therefore, this study aimed to investigate the mechanism of NFIB, and explore potential prognostic roles after platinum-doublet chemotherapy. In this study, we evaluated the mechanism of NFIB accelerating SCLC tumorigenesis. Furthermore, our findings revealed that NFIB inhibition modulates the activation of Notch signal to reduce SCLC chemosensitivity. Materials and Methods 1. Cell Culture and Transfection Analysis All cell lines were obtained from the American Type Culture Collection (ATCC) and grown in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-glutamine (Gibco). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. For lentiviral knockdown experiments, shRNA sequences targeting NFIB were produced by GeneChem (Shanghai, China). The lentiviral of NFIB overexpressing and a lentiviral vector expressing Notch1 NICD were also constructed by GeneChem (Shanghai, China). Then, cells were infected with lentiviral for 12 hours and selected by puromycin for 14 days. Knockdown was confirmed by qRT-PCR and western blotting. 2. Western blotting Whole-cell protein extracts were prepared in cold cell-lysis buffer (50mM Tris-HCl PH7.5, 150mM NaCl, 0.1% Sodium dodecyl sulfate, 1% Triton X-100, 0.5% Sodium deoxycholate, 5mM EDTA) supplemented with protease and phosphatase inhibitors. Proteins were quantified using BCA protein assay, resolved on SDS PAGE gels (4–20%) and transferred to nitrocellulose membranes. Alternatively, cells were counted and directly re-suspended in Laemmli buffer (1X). Protein samples were normalized to either GAPDH or actin-beta. A detailed list of antibodies used in the study can be found in table S1. 3. Real-time RT-PCR Total RNA was extracted in TRIzol (Invitrogen) and isolated according to the manufacturer’s protocol. cDNA was generated using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s protocol. qPCR experiments were run on a LightCycler®480 System (Roche). Data were normalized to GAPDH. A list of all the primers used for qRT–PCR can be found in table S2. 4. Cell viability and apoptosis assay For cell viability assay, 3000 cells were seeded in a 96-well plate overnight and treated with inhibitors or drugs as indicated times and concentration. Cell Counting Kit-8 (CCK-8) assays (Beyotime, China) were used and incubated at 37°C for 2 hours and measured at OD 450nm with the Tecan Sunrise™ system (Tecan, Switzerland). The optical density (OD) value was analysed by GraphPad Prism8.0 using non-linear regression. The equation log (inhibitor) versus response-variable slope was applied to plot the fitted dose-response curve. The IC50 was calculated as: Y=Bottom + (TopBottom)/ (1 + 10ˆ ((Log IC50-X) *HillSlope)). For apoptosis analysis, 1×10 6 cells were washed with cold PBS and then suspended in binding Buffer. Annexin V-FITC-PI dual-staining kit (BD Biosciences, 556547) was added 1:1 in suspensions and stained at RT for 15 minutes photophobic. Stained cells were screened by flow cytometry immediately. Cells were also harvested and using BIK, BAK, BAX and BCL-2 specific antibodies measured by western blotting. 5. Cisplatin and Inhibitor treatments Cells were seeded per well of a 96-well plate in RPMI-1640 media with 10% FBS. 10μl of drug solution was added per well the next day at the appropriate concentration and cell viability was assayed 48 hours later by the CCK8 assays. Cisplatin (Teva) was added from 0.1-20uM/ml. DBZ (Selleckchem, S2711) was added at a concentration of 10μM and tarextumab (OMP-59R5) at 100μg/ml. 6. ChIP-PCR experiments ChIP-PCR experiments was performed using techniques described previously with SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (CST, #9003) 19 . Briefly, cells cross-linked with 1% formaldehyde were resuspended in lysis buffer and sonicated to shear the DNA. Precleared lysates were incubated with either 10μg IgG or 2μg anti-NFI antibody (Abcam), followed by incubation with Protein A-Sepharose beads. Protein-DNA complexes were eluted, and the DNA was amplified using primers flanking putative NFI binding sites located upstream of the Notch1 transcription start site. Primer sequences flanking −317 to −417bp and −994 to −1105bp contained two putative NFI binding sites. The GAPDH promoter was used as the negative control. Input DNA was obtained from cells lysed after the sonication step. 7. Xenograft mice experiment H446 cells stable knockdown of NFIB were subcutaneously injected into the dorsal flank of the same athymic 5-week-old male BALB/c nude mice (n=5 for each group, Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 3 weeks, the mice were randomly allocated to six groups and treated with cisplatin (4mg/kg) and etoposide(4mg/kg)，tarextumab (20mg/kg), cisplatin and etoposide combined with tarextumab (20mg/kg) or vehicle PBS via intraperitoneal injection for indicated days (Figure 6C). The tumour volumes widest vertical width and longest diameter were measured before every injection day using a calliper. The tumor volume= (length × width 2 )/2. Finally, the tumor tissues from the xenograft mice were surgically removed, weighed and measured. The obtained tumor tissues were treated with immunohistochemistry staining to detect the protein levels of NFIB, Notch1 and Hes1. 8. Immunofluorescence (IF) and Immunohistochemistry (IHC) For IF cells were plated on cover slips, fixed in 4% PBS buffered formalin and pre-treated with 0.25% TritonX. Staining was performed for 30 minutes in a humidified chamber at RT. Images were taken by an inverted microscope fitted with a DM3000 (Leica). IHC stain was performed as previously described 10 . 9. Statistical analysis Statistics were calculated using Excel (Microsoft), Graph Pad Prism (STATCON) and SPSS (Armonk). We used two-sided Students t test. If normal distribution and similar variance in an experiment were not applicable, Kruskal-Wallis-Test was used. Error bars indicate standard error of the mean (SEM). Results 1. NFIB expression is correlated with high proliferation, low apoptosis and metastasis in SCLC Previous experiments have confirmed that NFIB is an oncogene in SCLC and accelerates SCLC initiation and progression. Fristly, to further clarify the specific evidence of NFIB participation in SCLC, we determined the amplifications of NFIB in human SCLC tissues and cells. The positive rate of NFIB staining was 80.7% (96/119) in SCLC tissues and 90.8% (59/65) in brain metastatic small cell carcinoma tissues, while NFIB was almost not expressed in carcinoid and LCNEC cells (Fig. 1 A, B and Figure S1 A). The positive rate of NFIB protein in brain metastatic small cell carcinoma tissues was significantly higher than that in primary SCLC tissues. High levels of NFIB was closely related to regional lymph node metastasis, distant metastasis and TNM stage (Table 1 ) and was also beneficial to intrapulmonary metastasis and vascular invasion in SCLC patients (Fig. 1 B). In addition, NFIB was expressed in SCLC and adjacent normal fresh tissues, and the expression level of NFIB was different among different SCLC patients. Among 24 patients, the expression level of NFIB mRNA in 21 cancer tissues was higher than that in adjacent normal tissues (Fig. 1 C). Moreover, NFIB expression was somewhat variability in different SCLC cell lines (Figure S1 B and C). Table 1 Correlations between NFIB expression and clinical/pathological features in SCLC. Clinicopathologic features Total, n % NFIB low NFIB high P n % n % value Gender Male 74 62.2 15 20.2 59 79.8 0.738 Female 45 37.8 8 17.8 37 82.2 Age ≤ 60 67 56.3 9 13.4 58 86.6 0.065 >60 52 43.7 14 26.9 38 73.1 Tobacco status Non-smoker 13 10.9 2 15.4 11 84.6 0.642 Former smoker 23 19.3 6 26.1 17 73.9 Active smoker 83 69.8 15 18.1 68 81.9 Histology type Pure SCLC 106 89.1 18 17 88 83 0.128 Mixed SCLC 13 10.9 5 38.5 8 61.5 Tumor size(cm) ≤ 3 75 63.1 17 22.7 58 77.3 0.336 >3 44 36.9 6 13.6 38 86.4 Syn Negative 17 14.3 15 88.2 2 11.8 < 0.001 Positive 102 85.7 8 7.8 94 92.2 CgA Negative 35 29.4 19 54.3 16 45.7 < 0.001 Positive 84 70.6 4 4.8 80 95.2 Ki-67 Negative 21 17.6 13 61.9 8 38.1 < 0.001 Positive 98 82.4 10 10.2 88 89.8 Lymph node metastasis Negative 61 51.3 17 27.9 44 72.1 0.020 Positive 58 48.7 6 10.3 52 89.7 Distant metastasis Negative 105 88.2 23 21.9 82 78.1 0.041 Positive 14 11.8 0 0 14 100 TNM staging Ⅰ 52 43.7 15 28.8 37 71.2 0.006 Ⅱ 33 27.7 6 18.2 27 81.8 Ⅲ 20 16.8 2 10 18 90 Ⅳ 14 11.8 0 0 14 100 Data were expressed as n (%). P < 0.05 was considered statistically significant. SCLC means small cell lung cancer. To determine the biological function of NFIB, we used shRNA to interfer NFIB in several human SCLC cell lines. While shRNA-mediated NFIB knockdown caused a dramatic increase in apoptosis and a corresponding decrease in proliferation and vice versa (Fig. 1 D-G). Notably, knockdown of NFIB mainly increased early apoptosis of tumor cells. Collectively, these data from both human patients and cells support an oncogenic role for NFIB in SCLC. 2. NFIB binds to sequences in BIK and BAK genes and represses pro-apoptotic factors To corroborate our findings of NFIB association with apoptosis, we analyzed proteins involved in this process by western blotting. Repression of NFIB increased levels of the pro-apoptotic factors, BIK and BAK, and reduced the ratio of BCL-2/ BAX (Fig. 2 A). Meanwhile, NFIB overexpression unsurprisingly led to different outcomes of related genes, although to a lesser extent than knockdown (Fig. 2 B). One interesting finding is that NFIB binds to the promoters of the pro-apoptotic factors confirmed by ChIP-PCR in H2227, which lacked NFIB amplification, by means of comparison with overexpression NFIB (Fig. 2 C). The rescue experiment in H209 also supports this result (Fig. 2 D-F). Taken together, NFIB inhibition in SCLC cells was sufficient to upregulate the expression of the pro-apoptotic factors, BIK and BAK, and promote apoptosis in these cells. Our data suggest that NFIB expression is integral to human SCLC cell line viability through its suppressing role of apoptosis. 3. Notch1 is required for the effect of NFIB on chemosensitivity We next examined whether NFIB can influence the acquisition of chemosensitivity in SCLC cells. A major factor in the dismal survival rates for SCLC is rapid relapse following standard-of chemotherapy (often cisplatin or carboplatin with etoposide) 20 . Therefore, we chose cisplatin and etoposide for the subsequent chemotherapy resistance trial. Wonderingly, NFIB knockdown tumor cell lines survived better than control cell lines in response to cisplatin and etoposide (Fig. 3 A and B). To further explore the specific mechanisms by which NFIB affects chemoresistance in SCLC cells, we applied GDS4794, GSE62021 and GSE60052 profiles to screen for genes associated with chemoresistance in SCLC (Fig. 3 C). We were surprised to find that NFIB is highly correlated with Notch genes which independently contributed to chemoresistance and intratumoral heterogeneity 21 , 22 . One of the putative NFI target gene, Hes1, was of particular interest because of its role as a critical transcriptional target of Notch signal pathway 10 . Hes1 has previously been shown to be important for chemotherapy resistance acquisition of SCLC cells and to be a negative prognostic factors for OS and PFS in SCLC patients 11 . Meanwhile, Notch signal pathway related genes, Notch1, Notch2, Notch4 and Hes1, was increased abundance following cisplatin treatment in mouse models of SCLC 23 . This model would suggest that Notch signaling activity is low in NFIB high SCLC cell lines and predict that decreased NFIB signaling would result in increased expression of Notch1 and Hes1. To examine this possibility, we first explored the transcriptional regulation of Hes1 and Notch1 by NFIB. Adenoviral transfer of shNFIB induced an increasing in Notch1, Notch2 and Hes1 expression levels (Fig. 3 D and E). Active NFIB caused a progressive reduction of Notch1, Notch2 and Hes1, reciprocal to the increase of NFIB (Fig. 3 E and I). These data suggest that NFIB inhibits Notch1, Notch2 and Hes1 gene expression in SCLC cells. To extend upon the finding that NFIB represses Notch1 expression, we wanted to further determine if the activity of Notch signal pathway was regulated by NFIB. Firstly, we treated H446-shNFIB with the γ-secretase inhibitor dibenzazepine (DBZ) 24 . Application of DBZ led to a significantly decreased expression of Notch1, Notch2 and Hes1 (Fig. 3 F). Moreover, inhibition of Notch activity was found to restore chemosensitivity in H446 shNFIB (Fig. 3 G and H). Meanwhile, stimulation of H2227-NFIB with active Notch1 intra-cellular domain(N1ICD) rescovered expression levels of Notch1, Notch2 and Hes1 (Fig. 3 I). Although overexpression of NFIB failed to change the chemosensitivity of SCLC cells, overexpression of Notch1 in H2227-NFIB could significantly reduce the drug sensitivity of cells to cisplain and etoposide (Fig. 3 J and K). The rescue experiment in H209 also proved this opinion (Figure S2A-F). Collectively, these results imply that NFIB impacts chemosensitivity by targeting Notch1. 4. NFIB directly represses Notch1 expression in SCLC To further characterize the transcriptional regulation of Notch1 by NFIB, we used IHC and IF to examine NFIB interacted with Notch1 in SCLC tissues and cells. It is worth noting that the expression of NFIB was markedly reduced in patients after chemotherapy, accompanied by a corresponding increase in expression levels of Notch1 and Hes1 (Fig. 4 A) and was negatively correlated with Notch1 and Hes1 in SCLC human tissues (Fig. 4 B). Significantly, NFIB, Notch1 and Hes1 showed co-localisation in SCLC cells (Fig. 4 C). Furthermore, we predicted that NFIB directly binds to the promoter of Notch1 by JASPAR. To confirm this prediction, we carried out ChIP analysis in H2227-NFIB cells using primers corresponding to the region of Notch1 promoter. Bands corresponding to the Notch1 promoter between − 368 to − 388bp and − 1038 to − 1058bp were clearly detected and enriched following immunoprecipitation with an NFIB antibody compared to rabbit IgG and the − 1038 to − 1058bp resulted in significant enrichment in binding, respectively(Fig. 4 D). Next, to test the transcriptional regulatory activity of sequences that bound the Notch1 promoter, we used a luciferase reporter system. The activity of an Notch1 promoter-luciferase reporter, or a mutant version in which NFIB consensus sites have been mutated (Mut), was determined in NFIB overexpression lines (H2227-NFIB). The immediate Notch1 promoter showed a significantly reduced level of luciferase activity with cotransfection of NFIB-pGL3 (Fig. 4 E). Together, these findings reveal that NFIB occupies the endogenous Notch1 promoter and can repress Notch1 promoter-driven gene expression. 5. NFIB knockdown accelerates the conversion of cell phenotype by targeting Notch activation Previous studies have shown that Notch1 and Hes1 were essential for maintaining the balance between NE and non-NE cells phenotype 9 , 25 and promoting phenotypic switching from NE to non-NE phenotype 11 . We hypothesized that the influence of NFIB on SCLC cells chemosensitivity may be related to cell phenotype transition which was regulated by Notch activation. As hypothesized, expression levels of NE markers, UCHL1, SYP, CALCA, CHGA and CHGB, were decreased in NFIB knockdown cells, instead the epithelial marker EPCAM was accordingly increased (Fig. 5 A, B and Figure S2C). Cell lines of SCLC cells transformed from typical NE SCLC floating cluster to adherent growth, further suggestive of a change in differentiation (Fig. 5 C). While this expression pattern was partially reversed after application of DBZ, expression levels of NE markers and EPCAM returned similar to levels before NFIB knockdown (Fig. 5 D-F). Similarly, this expression pattern is mitigated by stimulated with N1ICD (Fig. 5 G-I and Figure S2D). Meanwhile, NFIB expression was positively correlated with expression levels of SYP, CHGA and KI-67 in SCLC tissues (Figure S2G, Table 1 ). Collectively, these data further indicate that NFIB can inhibit the activation of Notch signal pathway and relate to the transition from NE to nonNE cell phenotype, therefore affect chemosensitivity of SCLC cells. 6. Inhibition of Notch activity reverses the effect of NFIB knockdown on chemosensitivity in vitro and vivo Additional chemotherapy resistance assays was applied to confirm the clinical significance of Notch signal pathway in the effect of NFIB on chemotherapy resistance through a combination of the Notch antagonist tarextumab and cisplatin in NFIB knockdown SCLC cells. Moreover, tarextumab markedly decreased protein levels of Notch1 and Notch2 confirmed by western blotting (Figure S3A). Importantly, tarextumab treatment was found to impair chemoresistance in these cells (Fig. 6 A and Figure S3B, C). Our experimental results are highly indicated that Notch1 is necessary for the affect of cisplatin resistance increasing induced by NFIB knockdown. Notably, the impact of NFIB overexpression on chemoresistance was altered upon Notch1 activation in H2227 cells (Fig. 6 B and Figure S3D, E). To further support the effect of NFIB on chemosensitivity and explore potential clinical applications, we established models in vivo. The nude mice injected with H446 cells after NFIB knockdown showed smaller volume of subcutaneous transplanted tumor and significantly lower sensitivity to cisplatin treatment than controls (NC). Moreover, combining tarextumab with cisplatin and etoposide strikingly decreased the tumor volumes of H446 shNFIB cells, whereas the administration of cisplatin and etoposide or tarextumab alone could not retard tumor growth (Fig. 6 C-E). Similar to in vitro results, immunohistochemical analysis results indicated a decreased levels of NFIB, Notch1 and Hes1 in the combination-treated group compared with other groups (Fig. 6 F). At the same time, multi-point puncture of subcutaneous tumors showed that the expression of NE genes was missing associated with increased expression of epithelial markers at large part of tissues after NFIB knockdown (Fig. 6 G and H). These findings suggested that knockdown NFIB may relieve the inhibition effect on Notch1 and initiate the transformation of cell phenotype to reduce chemosensitivity, but this process will not be reversed by overexpression of NFIB. Disscussion SCLC is a recalcitrant disease with an urgent need for more effective treatment approaches. Although SCLC is characterized by a relatively good response to first-line therapy, the majority of patients soon unfortunately relapse with resistant disease after an initial response. The first-line therapy for SCLC has not changed over several decades and there is no effective second-line therapy to date 26 – 28 . However, some more effective drug combinations, such as the combination of standard chemotherapy together with inhibitors targeting genes such as PD-L1, EZH2, CHK1 and ATR 29 – 31 , have recently been described. Importantly, regulation mechanisms underlying initial sensitivity and subsequent resistance of SCLC cells are not understood 32 , 33 . NFIB was previously identified as an oncogene in SCLC. Our data support that NFIB is frequently amplified in SCLC, especially in brain metastasis tissues. The expression of NFIB was highly related to regional lymph node metastasis, distant metastasis and TNM stage in SCLC patients, which is consistent with driver roles of NFIB for SCLC metastasis and high NFIB expression marks high-grade tumor populations both in a mouse model of SCLC and in human pNETs 18 . High NFIB expression was associated with migration and invasive behavior evidenced by our data (Figure S1 D and E). Furthermore, NFIB regulated pro-apoptotic factors to decreased apoptosis and promotion of proliferation. NFIB is important for brain development and neuronal migration, and several of the neuronal-associated NFIB-regulated genes 17 . In line with this, we found a consistent positive correlation between the expression of NFIB and NE differentiation markers in human SCLC tumors and cells. However, surprisingly, several researches have suggested that NFIB-expressing tumors responded better to treatment 23 . In the further study of the contradictory role of NFIB restricted to SCLC, we found that the effect of NFIB on tumor chemosensitivity was inconsistent with its effect on metastasis. To identify other essential proteins regulated by NFIB in chemoresistance, we performed a series of experiments and found its specific and strongest interaction with Notch1 and Hes1. In addition, this research found that Notch1 suppression blocked decreased chemosensitivity driven by NFIB and vice versa. The Notch signaling pathway mediates cell fate decisions and is tumor suppressive or oncogenic fuction depending on the context 4 , 34 , 35 . Numerous studies have identified Notch as a tumor suppressor and master regulator of neuroendocrine. However, emerging evidences suggest the critical role of Notch signal in developing SCLC chemoresistance by mediating the switch from NE to non-NE cells 11 . Our findings indicated that NFIB negatively regulated Notch1 and Hes1 expression by binding to Notch1 promoter region and provided new insights into the functional relationship between NFIB and the Notch pathway. As recent research also shows that NFI target genes include an effector of the Notch signaling pathway, HEY1, identified by ChIP data 36 . The role of Notch1 in SCLC is similar to a previous study, Notch1-low expression was associated with a better prognosis 37 . However, lake of Notch1 expression was an unfavorable prognostic factor in another study 38 . This study included only operable early stage SCLC cases, our study is limited since our cohort mainly consisted of cases with advanced stage of disease. Furthermore, ASCL1, a known target of Notch1 39 , as a master regulator of neuroendocrine cell fate was recently found to be essential for SCLC cell survival/viability 40–42 . However, our data showed that NFIB inhibited ASCL1 expression associated with Notch1 increasing (Fig.S4) but another ChIP-seq analysis showed that ASCL1 combined with NFIB in SCLC 42 . Pharmacological inhibition of Notch signaling partially rescued the effect of ASCL1 reduction (Fig.S4). Therefore, roles of ASCL1 in initiation and maintenance of NFIB expression maybe differ. The effect of NFIB overexpression on chemosensitivity is not as obvious as knockdown indicating that the chemosensitive effect of SCLC tumor cells may depend on activity of Notch1. In all, further studies need to focus on clarifying the mechanisms for NFIB regulating the other members of Notch signal pathway. SCLC tumors, which have few infiltrating stromal cells, can make their own diverse microenvironment 43 , 44 . Previous studies have shown that SCLC patients present with heterogeneous tumors, which at the time of diagnosis already consist of chemotherapy-sensitive and resistant tumor sub-populations 23 . Tumor heterogeneity in SCLC is pointed to as an important underlying mechanism of differential sensitivity to cisplatin treatment. This study suggests that NFIB high SCLC cells represent high invasion, migration and proliferation phenotypes, and are highly correlated with NE differentiation accompanied by high expression of NE gene and low expression of epithelial markers. NFIB knockdown activates Notch1 and may triggers the switch from NE-NFIB cells to non-NE Notch-active cells which are slow growing, consistent with a tumor suppressive role for Notch, but are also relatively chemoresistance, consistent with an oncogenic role. Together these results provide important insights into the crucial role of NFIB in tumor heterogeneity and its potential role in overcoming initial sensitivity and subsequent resistance to chemotherapy of SCLC. To highlight the clinical relevance, we tried to inhibit NFIB and Notch1 as a novel therapeutic avenue regarding the critical role of combination therapy in SCLC cancer development and chemoresistance. In phase 1b clinical trials, tarextumab treatment in combination with etoposide and cisplatin in patients with untreated extensive-stage SCLC, proved to be well-tolerated and showed a trend towards an improvement in overall and progression-free survival in SCLC patients with elevated levels of several Notch target genes, including Hes1 45 . Although tarextumab alone has a more negligible effect on inhibiting SCLC cell and its therapeutic effect is highly limited to the activity of Notch, combinations of tarextumab with cisplatin chemotherapy have displayed synergistic effects both in vitro and in vivo. Therefore, this study provided the potential therapeutic of the joint inhibition of NFIB and Notch1 for overcoming the acquired chemoresistance after initial chemosensitivity. Indeed, more perspective analyses would be necessary to verify the clinical relevance of the NFIB-Notch1-Hes1 axis in SCLC. Declarations Conflict of interest statement : The authors declare no conflict of interest. Acknowledgment : The authors thank all the pathology staffs and department of genetics, Harbin medical university for their commitment and significant effort. Ethics approval and consent to participate: The study was approved by the Ethics Committee to the Department of Pathology Harbin Medical University Cancer Hospital. Consent for publication : Written informed consent for publication was obtained from all participants. Competing interests : None. Funding : None. Authors' contributions ：Qin WX, Wang ZY, Deng SZ, Meng HX and Geng JS designed the research, and Qin WX, Wang ZY, Deng SZ and Qiu HL performed the research; Qin WX and Geng JS analyzed the data and wrote the paper. Acknowledgements ：The authors thank all the pathology staffs and department of genetics, Harbin medical university for their commitment and significant effort. Availability of data and material ：The authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary materials]. References Bernhardt EB, Jalal SI. Small Cell Lung Cancer. Cancer Treat Res. 2016;170:301–22. 10.1007/978-3-319-40389-2_14 . Bade BC, Dela Cruz CS, Lung Cancer. 2020: Epidemiology, Etiology, and Prevention. Clin Chest Med 41, 1–24, 10.1016/j.ccm.2019.10.001 (2020). 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Clin Cancer Res. 2015;21:2244–55. 10.1158/1078-0432.CCR-14-2958 . Saunders LR, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med. 2015;7:302ra136. 10.1126/scitranslmed.aac9459 . Zhdanovskaya N, et al. Targeting Notch to Maximize Chemotherapeutic Benefits: Rationale, Advanced Strategies, and Future Perspectives. Cancers (Basel). 2021;13. 10.3390/cancers13205106 . Böttger F, et al. Tumor Heterogeneity Underlies Differential Cisplatin Sensitivity in Mouse Models of Small-Cell Lung Cancer. Cell Rep. 2019;27:3345–3358e3344. 10.1016/j.celrep.2019.05.057 . Milano J, et al. Modulation of notch processing by gamma-secretase inhibitors causes intestinal goblet cell metaplasia and induction of genes known to specify gut secretory lineage differentiation. Toxicol Sci. 2004;82:341–58. 10.1093/toxsci/kfh254 . Ito T, et al. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development. 2000;127:3913–21. Farago AF, Keane FK. Current standards for clinical management of small cell lung cancer. Transl Lung Cancer Res. 2018;7:69–79. 10.21037/tlcr.2018.01.16 . Koinis F, Kotsakis A, Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years. Transl Lung Cancer Res. 2016;5:39–50. 10.3978/j.issn.2218-6751.2016.01.03 . Rossi A, Tay R, Chiramel J, Prelaj A, Califano R. Current and future therapeutic approaches for the treatment of small cell lung cancer. Expert Rev Anticancer Ther. 2018;18:473–86. 10.1080/14737140.2018.1453361 . Horn L, et al. First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer. N Engl J Med. 2018;379:2220–9. 10.1056/NEJMoa1809064 . Doerr F, et al. Targeting a non-oncogene addiction to the ATR/CHK1 axis for the treatment of small cell lung cancer. Sci Rep. 2017;7:15511. 10.1038/s41598-017-15840-5 . Gardner EE, et al. Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis. Cancer Cell. 2017;31:286–99. 10.1016/j.ccell.2017.01.006 . Ujhazy P, Lindwasser OW. Small cell lung cancer: updates and new concepts. Transl Lung Cancer Res. 2018;7:1–3. 10.21037/tlcr.2018.02.01 . Bunn PA Jr. et al. Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Translated into Improved Outcomes? J Thorac Oncol 11, 453–474, 10.1016/j.jtho.2016.01.012 (2016). Hori K, Sen A, Artavanis-Tsakonas S. Notch signaling at a glance. J Cell Sci. 2013;126:2135–40. 10.1242/jcs.127308 . Koch U, Radtke F. Notch and cancer: a double-edged sword. Cell Mol Life Sci. 2007;64:2746–62. 10.1007/s00018-007-7164-1 . Brun M, Jain S, Monckton EA, Godbout R. Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma. Neoplasia. 2018;20:1023–37. 10.1016/j.neo.2018.08.007 . Leonetti A, et al. Notch pathway in small-cell lung cancer: from preclinical evidence to therapeutic challenges. Cell Oncol. 2019;42:261–73. 10.1007/s13402-019-00441-3 . Kikuchi H, et al. Expression of Notch1 and Numb in small cell lung cancer. Oncotarget. 2017;8:10348–58. 10.18632/oncotarget.14411 . Sriuranpong V, et al. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol. 2002;22:3129–39. 10.1128/MCB.22.9.3129-3139.2002 . Augustyn A et al. ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers. Proceedings of the National Academy of Sciences 111, 14788–14793, 10.1073/pnas.1410419111 (2014). Jiang T, et al. Achaete-scute complex homologue 1 regulates tumor-initiating capacity in human small cell lung cancer. Cancer Res. 2009;69:845–54. 10.1158/0008-5472.CAN-08-2762 . Borromeo MD, et al. ASCL1 and NEUROD1 Reveal Heterogeneity in Pulmonary Neuroendocrine Tumors and Regulate Distinct Genetic Programs. Cell Rep. 2016;16:1259–72. 10.1016/j.celrep.2016.06.081 . Calbo J, et al. A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell. 2011;19:244–56. 10.1016/j.ccr.2010.12.021 . Williamson SC, et al. Vasculogenic mimicry in small cell lung cancer. Nat Commun. 2016;7:13322. 10.1038/ncomms13322 . Smith DC, et al. A phase 1 dose escalation and expansion study of Tarextumab (OMP-59R5) in patients with solid tumors. Invest New Drugs. 2019;37:722–30. 10.1007/s10637-018-0714-6 . Additional Declarations No competing interests reported. Supplementary Files supplementFigure.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3866018","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267364458,"identity":"0c776bc8-c4a6-4393-be9c-35bc08d7d645","order_by":0,"name":"Weixin Qin","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weixin","middleName":"","lastName":"Qin","suffix":""},{"id":267364459,"identity":"95c37e29-0712-43c8-b007-e96d0b312e53","order_by":1,"name":"Ziyan Wang","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ziyan","middleName":"","lastName":"Wang","suffix":""},{"id":267364460,"identity":"eee8d554-4135-4773-be2d-5ae3b6775223","order_by":2,"name":"Shuzhe Deng","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuzhe","middleName":"","lastName":"Deng","suffix":""},{"id":267364461,"identity":"eab704b0-822d-45f7-848c-d3892858197d","order_by":3,"name":"Huilei Qiu","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huilei","middleName":"","lastName":"Qiu","suffix":""},{"id":267364462,"identity":"e8f6a8dc-fcd8-4348-9c75-3a34b44ff324","order_by":4,"name":"Hongxue Meng","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongxue","middleName":"","lastName":"Meng","suffix":""},{"id":267364463,"identity":"2ed71908-ef24-41c7-95e3-fd5c9f20f4d8","order_by":5,"name":"Jingshu Geng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYBACxmYQ0QBhP2ZgOABmSRCrhdmYKC0QfRAtbNJEaWFu5z388ueOw3b90u3Xqgv33Ik2OMB88DYPg10ebofxpVnznjmcPHPOmbLbM549y91wgC3ZmochuRi3Fh4zY8a2w8kGN3LSbvMcOAzUwmMmzcNwILEBjxbDn0At9kAtxRAt/N8IaTF+wNt22M5AIv0YM9QWNoK2MPO2pSdI3MhhluY58Cx35mE2Y8s5Bsk4tRj2nzH++LPN2p5/RvrDzzwH7uT2HW9+eONNhR1uLQ0MbKBYACrgMYAIMYMIAxzqgUAeqOQDkLZnYGB/gFvZKBgFo2AUjGgAAIufXlgkVqODAAAAAElFTkSuQmCC","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jingshu","middleName":"","lastName":"Geng","suffix":""}],"badges":[],"createdAt":"2024-01-15 09:29:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3866018/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3866018/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49746732,"identity":"9cac38a5-feea-4003-b8db-a7312e313fca","added_by":"auto","created_at":"2024-01-17 11:10:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1049628,"visible":true,"origin":"","legend":"\u003cp\u003eNFIB reduces apoptosis and promotes proliferation and metastasis in SCLC.\u003c/p\u003e\n\u003cp\u003e(A) Quantification of NFIB expression as detected by IHC in human SCLC including primary tumors without metastasis (n=61), primary tumors with lung lymph node metastases (n=59) and brain metastases (n=65). IHC score, Low/negtive: 0-3, Medium: 4-8, High: 9-12. (B) NFIB staining showing intrapulmonary metastasis lesions within vascular and bronchiolar space of the lung. (C) Quantitative real-time PCR (qRT-PCR) analysis of NFIB expression (bottom) across a panel of primary SCLC and normal lung tissues. (D and E) PI/Annexin V staining and flow cytometry in SCLC cells following knockdown (D) or overexpression (E) NFIB transfection. (F and G) Growth curve of SCLC cells following knockdown (F) or overexpression (G) NFIB transfection using the CCK8 assay. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/63cf4f5b526bcb206456da76.png"},{"id":49747054,"identity":"4ce4150a-75f3-415d-bbaa-641c067e9db8","added_by":"auto","created_at":"2024-01-17 11:18:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":704626,"visible":true,"origin":"","legend":"\u003cp\u003eNFIB directly regulates pro-apoptotic factors in vitro.\u003c/p\u003e\n\u003cp\u003e(A and B) Western blotting was used to determine relative expression levels of multiple proteins of the BCL-2 family involved in the activation of apoptosis in NFIB knockdown and the anti-apoptosis in NFIB overexpression. GAPDH was used as a loading control. (C) ChIP-qPCR analysis of enrichment of NFIB binding within the BIK and BAK promoter. Precipitated DNAs were quantified with qRT-PCR for promoter regions of BIK and BAK gene. DNA cross-linked to protein in H2227 cells was immunoprecipitated with NFIB antibody followed by PCR amplification. Rabbit IgG antibody and GAPDH primers were used as negative controls. (D-F) Western blotting (D), CCK8 assay (E) and flow cytometry (F) were performed to detect role of NFIB on apoptosis in H209 treated with NFIB shRNA knock-down (H209 shNFIB) and control (H209 NC) and H209 shNFIB rescued by lentivirus to create stable overexpression of NFIB (H209 shNFIB-NFIB) .*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/827cf43fd6bc1022a8fdcde5.png"},{"id":49746730,"identity":"e3fa8ee5-ad1a-4daf-9fbf-45881d1a38f7","added_by":"auto","created_at":"2024-01-17 11:10:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":860185,"visible":true,"origin":"","legend":"\u003cp\u003eNFIB knockdown promotes chemoresistance by targeting Notch1 in vitro\u003c/p\u003e\n\u003cp\u003e(A and B) Relative cell viability of H446 cells with three different shRNA directed against NFIB compared with that of control cells (NC) treated with indicated concentrations of cisplatin (A) and etoposide (B) for 48 hours. (C) Expression levels of NFIB and the Notch family in microarray or RNA-seq datasets of SCLC tissues. GDS4794 (n=23), GSE62021 (n=25) and GSE60052 (n=86) datasets were analysed. Pearson correlation coefficients (r) and p-values are indicated. (D) Cell lysates from H446 treated with NFIB shRNA knock-down were analyzed by western blotting with antibodies against NFIB, Notch pathway genes (Notch1, Notch2, Hes1) and GAPDH. (E) Cell lysates from H209 NC, H209 shNFIB, H209 shNFIB NC and H209 shNFIB-NFIB were analyzed by western blotting with antibodies against NFIB, Notch pathway genes and GAPDH. (F-H) H446 NC and shNFIB were treated with DMSO control or DBZ, protein levels of NFIB, Notch pathway genes and GAPDH was measured through western blotting (F), and the relative cell viability of these cells treated with the indicated concentrations of cisplatin (G) and etoposide (H) for 48 hours was assessed by CCK8 assay. (I-K) H2227 stably overexpressing NFIB were transduced with a N1ICD retrovirus or an empty vector control to evaluate the protein level (I) and treated with different concentrations of cisplatin (J) and etoposide (K) for 48 hours, after which the cell viability was measured using CCK8 assay.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/65a2ae35510db565945aba00.png"},{"id":49747176,"identity":"8b1aa801-295c-4acd-a981-dbd8d4fa7a19","added_by":"auto","created_at":"2024-01-17 11:26:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1481507,"visible":true,"origin":"","legend":"\u003cp\u003eNFIB negatively regulates Notch1 expression in SCLC\u003c/p\u003e\n\u003cp\u003e(A) Representative NFIB, Notch1 and Hes1 staining of SCLC patients untreated and after chemotherapy. (B) The correlation between Notch1, Hes1 and NFIB in SCLC tissues assessed by IHC. Pearson correlation coefficients (r) and p-values are indicated. (C) Immunofluorescence on H446 parental cells grown on coverslips labeled with NFIB (green) , Notch1 (red) and Hes1 (red) antibody. DAPI used to stain cell nuclei. (D) Location of consensus NFIB binding sites and putative NFIB binding sequences identified upstream of the Notch1 transcription start site. Chromatin immunoprecipitation analysis showing NFIB binding to the Notch1 promoter. Rabbit IgG antibody and GAPDH primers were used as negative controls. (E) Luciferase reporter gene assay performed in H2227 cells stables for NFIB. A wild type construct containing a region of the Notch1 promoter containing the NFIB binding site (WT) and a mutant construct with the NFIB binding site mutated out (Mut). Mutation of the NFI-binding site at-1038 within the Notch1 promoter included two sites (Mut1 and Mut2). NFIB pCAGIG was added to envalue the influence of luciferase activity in above types. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/bb5f1aa77b472ee694bdd78a.png"},{"id":49746734,"identity":"39923eb3-1c26-4320-9c01-ac09834f4d27","added_by":"auto","created_at":"2024-01-17 11:10:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1821345,"visible":true,"origin":"","legend":"\u003cp\u003eNFIB knockdown activate Notch1 and promote the growth of non-NE SCLC cells.\u003c/p\u003e\n\u003cp\u003e(A-B) Western blotting (A) and qRT-PCR (B) for Notch pathway genes and NE genes in H446 cells transduced with NFIB shRNA.***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0005. (C) Representative images of H446 after NFIB knockdown. Scale bars, 50μm. (D-E) Western blotting (D) and qRT-PCR (E) for Notch pathway genes and NE genes in H446 shNFIB cells treatced with DBZ. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. H446 NC; \u003csup\u003e# \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. H446 shNFIB+DMSO. (F) Representative images of H446 shNFIB after DBZ treatment. (G-I) Western blotting (G), qRT-PCR (H) and images (I) of H2227-NFIB after N1ICD transduction. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. H2227 NC; \u003csup\u003e#\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. H2227-NFIB NC.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/7e90f1a9f78c0ef5f097f59d.png"},{"id":49746736,"identity":"f12ed040-b7ef-4dff-bc58-fcd0953ffc41","added_by":"auto","created_at":"2024-01-17 11:10:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3401614,"visible":true,"origin":"","legend":"\u003cp\u003eA combination of chemotherapy and Notch blockade improves SCLC chemosensitivity lacked of NFIB in vitro and vivo.\u003c/p\u003e\n\u003cp\u003e(A) Relative cell viability of H446 shNFIB cells pretreated with tarextumab (tarex) combined with the indicated concentrations of cisplatin for 48 hours.(B) Relative cell viability of H2227-NFIB stable overexpression N1ICD cells pretreated with tarextumab combined with the indicated concentrations of cisplatin for 48 hours.(C) Xenografts in nude mice by inoculating H446 cells with NFIB knockdown or empty vector (NC) (n=5/group). The administration of drug dose and time was recorded, representative photographs of tumours excised from mices on 40 days are shown.(D) The growth curve showed changes in tumour volume in mice from different groups.(E) Tumour volume of tumours excised from mices in each group.(F) Representative H\u0026amp;E staining and IHC images of NFIB and Hes1 in excised tumour tissues. Scale bars=25μm.(G) qRT-PCR analysis and IHC staining of NE genes of tumours excised from mices in each group. ns, Not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0005.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/a190dd6f2f2773fffc58bb21.png"},{"id":51203429,"identity":"27a351df-250b-496d-b83e-769c13584661","added_by":"auto","created_at":"2024-02-16 00:15:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3800478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/9eeead43-fc3b-4adb-a2d5-951d4bc75c13.pdf"},{"id":49746735,"identity":"1ced3f12-4139-4b55-9952-f7508f434c50","added_by":"auto","created_at":"2024-01-17 11:10:46","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1078673,"visible":true,"origin":"","legend":"","description":"","filename":"supplementFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866018/v1/157b422972f38996208e609b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"NFIB controls chemosensitivity in small cell lung cancer by suppressing Notch signaling activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSmall cell lung cancer(SCLC) is the most aggressive subtype of lung cancer, which accounts for 13% of all lung cancers, characterized by rapid growth and early metastatic spread\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. SCLC patients typically present with initial chemosensitivity, followed by the rapid emergence of chemoresistance, the 5-year overall survival (OS) is \u0026lt;\u0026thinsp;10%\u003csup\u003e2\u003c/sup\u003e. There has been limited improvement in first-line SCLC treatment in the past 30 years and no targeted therapies are currently available\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, mechanisms underlying metastases and rapidly resistance of SCLC cells are not understood, and SCLC remains a recalcitrant cancer.\u003c/p\u003e \u003cp\u003eNotch singling plays an important role in cell-fate decisions in a variety of tissues\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Activation of Notch family members potently suppresses in SCLC mouse models during lung development intrinsically to NE cells and also can be protumorigenic through produceing non-NE cells that are more chemoresistant\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Activation of the Notch pathway results in a NE to non-NE fate switch occurred in 10\u0026ndash;50% of NE cells in SCLC and coincided with the induction of REST, a transcriptional repressor that inhibits NE gene expression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. One gene that is regulated by the Notch1 is hairy/ enhancer of split (Hes1), a basic helix-loop-helix transcription factor, which has been described as a negative prognostic factor for overall and progression-free survival in SCLC tumors. In addition, ASCL1, a transcription factor that promotes neuroendocrine transcriptional programs, appears to be a direct downstream target of Notch singling\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNFIB belongs to the nuclear factor I (NFI) family of transcription factors which play important roles during lung and brain development and have been associated with developmental abnormalities in humans\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The NFI family consists of four transcription factors in humans and most vertebrates: NFIA, NFIB, NFIC, and NFIX, all have a homologous DNA binding domain and function in adenoviral DNA replication and in the regulation of transcription of a large variety of cellular and viral genes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Recent studies have defined NFIB as an oncogene in SCLC\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Through NFIB promotes metastasis through a widespread increase in chromatin accessibility and changes the metastatic profile in a Mouse Model of SCLC, increased expression of the NFIB was identified as a pro-metastatic switch\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. While NFIB upregulation may represent critical to metastasis in a subset of human SCLC, the molecular mechanism of NFIB in tumorigenic process and possible associations with sensitivity to chemotherapy, prognosis, and clinical characteristics of SCLC remained elusive. Therefore, this study aimed to investigate the mechanism of NFIB, and explore potential prognostic roles after platinum-doublet chemotherapy. In this study, we evaluated the mechanism of NFIB accelerating SCLC tumorigenesis. Furthermore, our findings revealed that NFIB inhibition modulates the activation of Notch signal to reduce SCLC chemosensitivity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Cell Culture and Transfection Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cell lines were obtained from the American Type Culture Collection (ATCC) and grown in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-glutamine (Gibco). Cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO2 and 95% air. For lentiviral knockdown experiments, shRNA sequences targeting NFIB were produced by GeneChem (Shanghai, China). The lentiviral of NFIB overexpressing and a lentiviral vector expressing Notch1 NICD were also constructed by GeneChem (Shanghai, China). Then, cells were infected with lentiviral for 12 hours and selected by puromycin for 14 days. Knockdown was confirmed by qRT-PCR and western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell protein extracts were prepared in cold cell-lysis buffer (50mM Tris-HCl PH7.5, 150mM NaCl, 0.1% Sodium dodecyl sulfate, 1% Triton X-100, 0.5% Sodium deoxycholate, 5mM EDTA) supplemented with protease and phosphatase inhibitors. Proteins were quantified using BCA protein assay, resolved on SDS PAGE gels (4\u0026ndash;20%) and transferred to nitrocellulose membranes. Alternatively, cells were counted and directly re-suspended in Laemmli buffer (1X). Protein samples were normalized to either GAPDH or actin-beta. A detailed list of antibodies used in the study can be found in table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Real-time RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted in TRIzol (Invitrogen) and isolated according to the manufacturer\u0026rsquo;s protocol. cDNA was generated using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer\u0026rsquo;s protocol. qPCR experiments were run on a LightCycler\u0026reg;480 System (Roche). Data were normalized to GAPDH. A list of all the primers used for qRT\u0026ndash;PCR can be found in table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Cell viability and apoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell viability assay, 3000 cells were seeded in a 96-well plate overnight and treated with inhibitors or drugs as indicated times and concentration. Cell Counting Kit-8 (CCK-8) assays (Beyotime, China) were used and incubated at 37\u0026deg;C for 2 hours and measured at OD 450nm with the Tecan Sunrise\u0026trade; system (Tecan, Switzerland). The optical density (OD) value was analysed by GraphPad Prism8.0 using non-linear regression. The equation log (inhibitor) versus response-variable slope was applied to plot the fitted dose-response curve. The IC50 was calculated as: Y=Bottom + (TopBottom)/ (1 + 10\u0026circ; ((Log IC50-X) *HillSlope)).\u003c/p\u003e\n\u003cp\u003eFor apoptosis analysis, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were washed with cold PBS and then suspended in binding Buffer. Annexin V-FITC-PI dual-staining kit (BD Biosciences, 556547) was added 1:1 in suspensions and stained at RT for 15 minutes photophobic. Stained cells were screened by flow cytometry immediately. Cells were also harvested and using BIK, BAK, BAX and BCL-2 specific antibodies measured by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Cisplatin and Inhibitor treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded per well of a 96-well plate in RPMI-1640 media with 10% FBS. 10\u0026mu;l of drug solution was added per well the next day at the appropriate concentration and cell viability was assayed 48 hours later by the CCK8 assays. Cisplatin (Teva) was added from 0.1-20uM/ml. DBZ (Selleckchem, S2711) was added at a concentration of 10\u0026mu;M and tarextumab (OMP-59R5) at 100\u0026mu;g/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. ChIP-PCR experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP-PCR experiments was performed using techniques described previously with SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit (Magnetic Beads) (CST, #9003)\u003csup\u003e19\u003c/sup\u003e. Briefly, cells cross-linked with 1% formaldehyde were resuspended in lysis buffer and sonicated to shear the DNA. Precleared lysates were incubated with either 10\u0026mu;g IgG or 2\u0026mu;g anti-NFI antibody (Abcam), followed by incubation with Protein A-Sepharose beads. Protein-DNA complexes were eluted, and the DNA was amplified using primers flanking putative NFI binding sites located upstream of the Notch1 transcription start site. Primer sequences flanking \u0026minus;317 to \u0026minus;417bp and \u0026minus;994 to \u0026minus;1105bp contained two putative NFI binding sites. The GAPDH promoter was used as the negative control. Input DNA was obtained from cells lysed after the sonication step.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Xenograft mice experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH446 cells stable knockdown of NFIB were subcutaneously injected into the dorsal flank of the same athymic 5-week-old male BALB/c nude mice (n=5 for each group, Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 3 weeks, the mice were randomly allocated to six groups and treated with cisplatin (4mg/kg) and etoposide(4mg/kg)，tarextumab (20mg/kg), cisplatin and etoposide combined with tarextumab (20mg/kg) or vehicle PBS via intraperitoneal injection for indicated days (Figure 6C). The tumour volumes widest vertical width and longest diameter were measured before every injection day using a calliper. The tumor volume= (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. Finally, the tumor tissues from the xenograft mice were surgically removed, weighed and measured. The obtained tumor tissues were treated with immunohistochemistry staining to detect the protein levels of NFIB, Notch1 and Hes1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Immunofluorescence (IF) and Immunohistochemistry (IHC)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor IF cells were plated on cover slips, fixed in 4% PBS buffered formalin and pre-treated with 0.25% TritonX. Staining was performed for 30 minutes in a humidified chamber at RT. Images were taken by an inverted microscope fitted with a DM3000 (Leica). IHC stain was performed as previously described\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistics were calculated using Excel (Microsoft), Graph Pad Prism (STATCON) and SPSS (Armonk). We used two-sided Students t test. If normal distribution and similar variance in an experiment were not applicable, Kruskal-Wallis-Test was used. Error bars indicate standard error of the mean (SEM).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. NFIB expression is correlated with high proliferation, low apoptosis and metastasis in SCLC\u003c/p\u003e \u003cp\u003ePrevious experiments have confirmed that NFIB is an oncogene in SCLC and accelerates SCLC initiation and progression. Fristly, to further clarify the specific evidence of NFIB participation in SCLC, we determined the amplifications of NFIB in human SCLC tissues and cells. The positive rate of NFIB staining was 80.7% (96/119) in SCLC tissues and 90.8% (59/65) in brain metastatic small cell carcinoma tissues, while NFIB was almost not expressed in carcinoid and LCNEC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The positive rate of NFIB protein in brain metastatic small cell carcinoma tissues was significantly higher than that in primary SCLC tissues. High levels of NFIB was closely related to regional lymph node metastasis, distant metastasis and TNM stage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and was also beneficial to intrapulmonary metastasis and vascular invasion in SCLC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, NFIB was expressed in SCLC and adjacent normal fresh tissues, and the expression level of NFIB was different among different SCLC patients. Among 24 patients, the expression level of NFIB mRNA in 21 cancer tissues was higher than that in adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, NFIB expression was somewhat variability in different SCLC cell lines (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between NFIB expression and clinical/pathological features in SCLC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eClinicopathologic\u003c/p\u003e \u003cp\u003efeatures\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal, n\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNFIB low\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eNFIB high\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003evalue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e79.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.738\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e82.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e86.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e73.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTobacco status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon-smoker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e84.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.642\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFormer smoker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e73.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive smoker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e81.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistology type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePure SCLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed SCLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size(cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e77.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.336\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e86.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e92.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCgA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e45.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKi-67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e38.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e82.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e89.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e72.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.020\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e89.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e78.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.041\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM staging\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e71.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e81.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅣ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eData were expressed as n (%). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eSCLC means small cell lung cancer.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo determine the biological function of NFIB, we used shRNA to interfer NFIB in several human SCLC cell lines. While shRNA-mediated NFIB knockdown caused a dramatic increase in apoptosis and a corresponding decrease in proliferation and vice versa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Notably, knockdown of NFIB mainly increased early apoptosis of tumor cells. Collectively, these data from both human patients and cells support an oncogenic role for NFIB in SCLC.\u003c/p\u003e \u003cp\u003e2. NFIB binds to sequences in BIK and BAK genes and represses pro-apoptotic factors\u003c/p\u003e \u003cp\u003eTo corroborate our findings of NFIB association with apoptosis, we analyzed proteins involved in this process by western blotting. Repression of NFIB increased levels of the pro-apoptotic factors, BIK and BAK, and reduced the ratio of BCL-2/ BAX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Meanwhile, NFIB overexpression unsurprisingly led to different outcomes of related genes, although to a lesser extent than knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). One interesting finding is that NFIB binds to the promoters of the pro-apoptotic factors confirmed by ChIP-PCR in H2227, which lacked NFIB amplification, by means of comparison with overexpression NFIB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The rescue experiment in H209 also supports this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). Taken together, NFIB inhibition in SCLC cells was sufficient to upregulate the expression of the pro-apoptotic factors, BIK and BAK, and promote apoptosis in these cells. Our data suggest that NFIB expression is integral to human SCLC cell line viability through its suppressing role of apoptosis.\u003c/p\u003e\u003cp\u003e3. Notch1 is required for the effect of NFIB on chemosensitivity\u003c/p\u003e\u003cp\u003eWe next examined whether NFIB can influence the acquisition of chemosensitivity in SCLC cells. A major factor in the dismal survival rates for SCLC is rapid relapse following standard-of chemotherapy (often cisplatin or carboplatin with etoposide)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, we chose cisplatin and etoposide for the subsequent chemotherapy resistance trial. Wonderingly, NFIB knockdown tumor cell lines survived better than control cell lines in response to cisplatin and etoposide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). To further explore the specific mechanisms by which NFIB affects chemoresistance in SCLC cells, we applied GDS4794, GSE62021 and GSE60052 profiles to screen for genes associated with chemoresistance in SCLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We were surprised to find that NFIB is highly correlated with Notch genes which independently contributed to chemoresistance and intratumoral heterogeneity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. One of the putative NFI target gene, Hes1, was of particular interest because of its role as a critical transcriptional target of Notch signal pathway\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Hes1 has previously been shown to be important for chemotherapy resistance acquisition of SCLC cells and to be a negative prognostic factors for OS and PFS in SCLC patients\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Meanwhile, Notch signal pathway related genes, Notch1, Notch2, Notch4 and Hes1, was increased abundance following cisplatin treatment in mouse models of SCLC\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This model would suggest that Notch signaling activity is low in NFIB\u003csup\u003ehigh\u003c/sup\u003e SCLC cell lines and predict that decreased NFIB signaling would result in increased expression of Notch1 and Hes1. To examine this possibility, we first explored the transcriptional regulation of Hes1 and Notch1 by NFIB. Adenoviral transfer of shNFIB induced an increasing in Notch1, Notch2 and Hes1 expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). Active NFIB caused a progressive reduction of Notch1, Notch2 and Hes1, reciprocal to the increase of NFIB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and I). These data suggest that NFIB inhibits Notch1, Notch2 and Hes1 gene expression in SCLC cells.\u003c/p\u003e \u003cp\u003eTo extend upon the finding that NFIB represses Notch1 expression, we wanted to further determine if the activity of Notch signal pathway was regulated by NFIB. Firstly, we treated H446-shNFIB with the γ-secretase inhibitor dibenzazepine (DBZ)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Application of DBZ led to a significantly decreased expression of Notch1, Notch2 and Hes1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Moreover, inhibition of Notch activity was found to restore chemosensitivity in H446 shNFIB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H). Meanwhile, stimulation of H2227-NFIB with active Notch1 intra-cellular domain(N1ICD) rescovered expression levels of Notch1, Notch2 and Hes1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Although overexpression of NFIB failed to change the chemosensitivity of SCLC cells, overexpression of Notch1 in H2227-NFIB could significantly reduce the drug sensitivity of cells to cisplain and etoposide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ and K). The rescue experiment in H209 also proved this opinion (Figure S2A-F). Collectively, these results imply that NFIB impacts chemosensitivity by targeting Notch1.\u003c/p\u003e \u003cp\u003e4. NFIB directly represses Notch1 expression in SCLC\u003c/p\u003e\u003cp\u003eTo further characterize the transcriptional regulation of Notch1 by NFIB, we used IHC and IF to examine NFIB interacted with Notch1 in SCLC tissues and cells. It is worth noting that the expression of NFIB was markedly reduced in patients after chemotherapy, accompanied by a corresponding increase in expression levels of Notch1 and Hes1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and was negatively correlated with Notch1 and Hes1 in SCLC human tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Significantly, NFIB, Notch1 and Hes1 showed co-localisation in SCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, we predicted that NFIB directly binds to the promoter of Notch1 by JASPAR. To confirm this prediction, we carried out ChIP analysis in H2227-NFIB cells using primers corresponding to the region of Notch1 promoter. Bands corresponding to the Notch1 promoter between \u0026minus;\u0026thinsp;368 to \u0026minus;\u0026thinsp;388bp and \u0026minus;\u0026thinsp;1038 to \u0026minus;\u0026thinsp;1058bp were clearly detected and enriched following immunoprecipitation with an NFIB antibody compared to rabbit IgG and the \u0026minus;\u0026thinsp;1038 to \u0026minus;\u0026thinsp;1058bp resulted in significant enrichment in binding, respectively(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to test the transcriptional regulatory activity of sequences that bound the Notch1 promoter, we used a luciferase reporter system. The activity of an Notch1 promoter-luciferase reporter, or a mutant version in which NFIB consensus sites have been mutated (Mut), was determined in NFIB overexpression lines (H2227-NFIB). The immediate Notch1 promoter showed a significantly reduced level of luciferase activity with cotransfection of NFIB-pGL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Together, these findings reveal that NFIB occupies the endogenous Notch1 promoter and can repress Notch1 promoter-driven gene expression.\u003c/p\u003e \u003cp\u003e5. NFIB knockdown accelerates the conversion of cell phenotype by targeting Notch activation\u003c/p\u003e \u003cp\u003ePrevious studies have shown that Notch1 and Hes1 were essential for maintaining the balance between NE and non-NE cells phenotype\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and promoting phenotypic switching from NE to non-NE phenotype\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We hypothesized that the influence of NFIB on SCLC cells chemosensitivity may be related to cell phenotype transition which was regulated by Notch activation. As hypothesized, expression levels of NE markers, UCHL1, SYP, CALCA, CHGA and CHGB, were decreased in NFIB knockdown cells, instead the epithelial marker EPCAM was accordingly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and Figure S2C). Cell lines of SCLC cells transformed from typical NE SCLC floating cluster to adherent growth, further suggestive of a change in differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). While this expression pattern was partially reversed after application of DBZ, expression levels of NE markers and EPCAM returned similar to levels before NFIB knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). Similarly, this expression pattern is mitigated by stimulated with N1ICD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I and Figure S2D). Meanwhile, NFIB expression was positively correlated with expression levels of SYP, CHGA and KI-67 in SCLC tissues (Figure S2G, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Collectively, these data further indicate that NFIB can inhibit the activation of Notch signal pathway and relate to the transition from NE to nonNE cell phenotype, therefore affect chemosensitivity of SCLC cells.\u003c/p\u003e \u003cp\u003e6. Inhibition of Notch activity reverses the effect of NFIB knockdown on chemosensitivity in vitro and vivo\u003c/p\u003e \u003cp\u003eAdditional chemotherapy resistance assays was applied to confirm the clinical significance of Notch signal pathway in the effect of NFIB on chemotherapy resistance through a combination of the Notch antagonist tarextumab and cisplatin in NFIB knockdown SCLC cells. Moreover, tarextumab markedly decreased protein levels of Notch1 and Notch2 confirmed by western blotting (Figure S3A). Importantly, tarextumab treatment was found to impair chemoresistance in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Figure S3B, C). Our experimental results are highly indicated that Notch1 is necessary for the affect of cisplatin resistance increasing induced by NFIB knockdown. Notably, the impact of NFIB overexpression on chemoresistance was altered upon Notch1 activation in H2227 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Figure S3D, E).\u003c/p\u003e \u003cp\u003eTo further support the effect of NFIB on chemosensitivity and explore potential clinical applications, we established models in vivo. The nude mice injected with H446 cells after NFIB knockdown showed smaller volume of subcutaneous transplanted tumor and significantly lower sensitivity to cisplatin treatment than controls (NC). Moreover, combining tarextumab with cisplatin and etoposide strikingly decreased the tumor volumes of H446 shNFIB cells, whereas the administration of cisplatin and etoposide or tarextumab alone could not retard tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E). Similar to in vitro results, immunohistochemical analysis results indicated a decreased levels of NFIB, Notch1 and Hes1 in the combination-treated group compared with other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). At the same time, multi-point puncture of subcutaneous tumors showed that the expression of NE genes was missing associated with increased expression of epithelial markers at large part of tissues after NFIB knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and H). These findings suggested that knockdown NFIB may relieve the inhibition effect on Notch1 and initiate the transformation of cell phenotype to reduce chemosensitivity, but this process will not be reversed by overexpression of NFIB.\u003c/p\u003e"},{"header":"Disscussion","content":"\u003cp\u003eSCLC is a recalcitrant disease with an urgent need for more effective treatment approaches. Although SCLC is characterized by a relatively good response to first-line therapy, the majority of patients soon unfortunately relapse with resistant disease after an initial response. The first-line therapy for SCLC has not changed over several decades and there is no effective second-line therapy to date\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, some more effective drug combinations, such as the combination of standard chemotherapy together with inhibitors targeting genes such as PD-L1, EZH2, CHK1 and ATR\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, have recently been described. Importantly, regulation mechanisms underlying initial sensitivity and subsequent resistance of SCLC cells are not understood\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNFIB was previously identified as an oncogene in SCLC. Our data support that NFIB is frequently amplified in SCLC, especially in brain metastasis tissues. The expression of NFIB was highly related to regional lymph node metastasis, distant metastasis and TNM stage in SCLC patients, which is consistent with driver roles of NFIB for SCLC metastasis and high NFIB expression marks high-grade tumor populations both in a mouse model of SCLC and in human pNETs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. High NFIB expression was associated with migration and invasive behavior evidenced by our data (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD and E). Furthermore, NFIB regulated pro-apoptotic factors to decreased apoptosis and promotion of proliferation. NFIB is important for brain development and neuronal migration, and several of the neuronal-associated NFIB-regulated genes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In line with this, we found a consistent positive correlation between the expression of NFIB and NE differentiation markers in human SCLC tumors and cells.\u003c/p\u003e \u003cp\u003eHowever, surprisingly, several researches have suggested that NFIB-expressing tumors responded better to treatment\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the further study of the contradictory role of NFIB restricted to SCLC, we found that the effect of NFIB on tumor chemosensitivity was inconsistent with its effect on metastasis. To identify other essential proteins regulated by NFIB in chemoresistance, we performed a series of experiments and found its specific and strongest interaction with Notch1 and Hes1. In addition, this research found that Notch1 suppression blocked decreased chemosensitivity driven by NFIB and vice versa. The Notch signaling pathway mediates cell fate decisions and is tumor suppressive or oncogenic fuction depending on the context\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Numerous studies have identified Notch as a tumor suppressor and master regulator of neuroendocrine. However, emerging evidences suggest the critical role of Notch signal in developing SCLC chemoresistance by mediating the switch from NE to non-NE cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Our findings indicated that NFIB negatively regulated Notch1 and Hes1 expression by binding to Notch1 promoter region and provided new insights into the functional relationship between NFIB and the Notch pathway. As recent research also shows that NFI target genes include an effector of the Notch signaling pathway, HEY1, identified by ChIP data\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The role of Notch1 in SCLC is similar to a previous study, Notch1-low expression was associated with a better prognosis\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, lake of Notch1 expression was an unfavorable prognostic factor in another study\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This study included only operable early stage SCLC cases, our study is limited since our cohort mainly consisted of cases with advanced stage of disease. Furthermore, ASCL1, a known target of Notch1\u003csup\u003e39\u003c/sup\u003e, as a master regulator of neuroendocrine cell fate was recently found to be essential for SCLC cell survival/viability\u003csup\u003e40\u0026ndash;42\u003c/sup\u003e. However, our data showed that NFIB inhibited ASCL1 expression associated with Notch1 increasing (Fig.S4) but another ChIP-seq analysis showed that ASCL1 combined with NFIB in SCLC\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Pharmacological inhibition of Notch signaling partially rescued the effect of ASCL1 reduction (Fig.S4). Therefore, roles of ASCL1 in initiation and maintenance of NFIB expression maybe differ. The effect of NFIB overexpression on chemosensitivity is not as obvious as knockdown indicating that the chemosensitive effect of SCLC tumor cells may depend on activity of Notch1. In all, further studies need to focus on clarifying the mechanisms for NFIB regulating the other members of Notch signal pathway.\u003c/p\u003e \u003cp\u003eSCLC tumors, which have few infiltrating stromal cells, can make their own diverse microenvironment\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that SCLC patients present with heterogeneous tumors, which at the time of diagnosis already consist of chemotherapy-sensitive and resistant tumor sub-populations\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Tumor heterogeneity in SCLC is pointed to as an important underlying mechanism of differential sensitivity to cisplatin treatment. This study suggests that NFIB\u003csup\u003ehigh\u003c/sup\u003e SCLC cells represent high invasion, migration and proliferation phenotypes, and are highly correlated with NE differentiation accompanied by high expression of NE gene and low expression of epithelial markers. NFIB knockdown activates Notch1 and may triggers the switch from NE-NFIB cells to non-NE Notch-active cells which are slow growing, consistent with a tumor suppressive role for Notch, but are also relatively chemoresistance, consistent with an oncogenic role. Together these results provide important insights into the crucial role of NFIB in tumor heterogeneity and its potential role in overcoming initial sensitivity and subsequent resistance to chemotherapy of SCLC.\u003c/p\u003e \u003cp\u003eTo highlight the clinical relevance, we tried to inhibit NFIB and Notch1 as a novel therapeutic avenue regarding the critical role of combination therapy in SCLC cancer development and chemoresistance. In phase 1b clinical trials, tarextumab treatment in combination with etoposide and cisplatin in patients with untreated extensive-stage SCLC, proved to be well-tolerated and showed a trend towards an improvement in overall and progression-free survival in SCLC patients with elevated levels of several Notch target genes, including Hes1\u003csup\u003e45\u003c/sup\u003e. Although tarextumab alone has a more negligible effect on inhibiting SCLC cell and its therapeutic effect is highly limited to the activity of Notch, combinations of tarextumab with cisplatin chemotherapy have displayed synergistic effects both in vitro and in vivo. Therefore, this study provided the potential therapeutic of the joint inhibition of NFIB and Notch1 for overcoming the acquired chemoresistance after initial chemosensitivity. Indeed, more perspective analyses would be necessary to verify the clinical relevance of the NFIB-Notch1-Hes1 axis in SCLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e: The authors thank all the pathology staffs and department of genetics, Harbin medical university for their commitment and significant effort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e The study was approved by the Ethics Committee to the Department of Pathology Harbin Medical University Cancer Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Written informed consent for publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e：Qin WX, Wang ZY, Deng SZ, Meng HX and Geng JS designed the research, and Qin WX, Wang ZY, Deng SZ and Qiu HL performed the research; Qin WX and Geng JS analyzed the data and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e：The authors thank all the pathology staffs and department of genetics, Harbin medical university for their commitment and significant effort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e：The authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary materials].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBernhardt EB, Jalal SI. 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Invest New Drugs. 2019;37:722\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10637-018-0714-6\u003c/span\u003e\u003cspan address=\"10.1007/s10637-018-0714-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NFIB, Notch, chemosensitivity, heterogeneity, SCLC","lastPublishedDoi":"10.21203/rs.3.rs-3866018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3866018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eSmall cell lung cancer(SCLC) is a highly aggressive malignant tumor type, characterized by universal acquired therapeutic resistance during rapidly recurrence. However, we have a poor understanding of the mechanisms underlying development of resistance. NFIB is a bona fide oncogene in SCLC with effects on proliferation, invasion, and apoptosis inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe expression of NFIB and related genes was evaluated in SCLC cells and tissue specimens, by western blot, RT-PCR immunofluorescence and immunohistochemistry. The relationship between genes was verified by ChIP-PCR experiments. CCK8 assays was used for drug resistance experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eNFIB is highly correlated with NE markers and may be participated in the generating of tumor heterogeneity mediated in part by Notch1. The suppressive effect of NFIB on Notch1 is relieved and led to NE gene inhibiting when SCLC cells lack of NFIB. These cells are slow growing and also relatively chemoresistant. Importantly, Notch blockade in combination with chemotherapy alleviates the formation of intratumoral heterogeneity and enhances chemosensitivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eNFIB can be both tumor-pro-tumorigenic and chemosensitizing in SCLC. NFIB knockdown results in endogenous activation of the Notch pathway. These results uncover the dual character of oncogene as NFIB in SCLC and offer perspectives for efficacious combination therapies that might also hold promise for treating human SCLC.\u003c/p\u003e","manuscriptTitle":"NFIB controls chemosensitivity in small cell lung cancer by suppressing Notch signaling activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 11:10:41","doi":"10.21203/rs.3.rs-3866018/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f463f3ee-34f2-4251-99e9-9b03f5c996e9","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-16T00:07:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 11:10:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3866018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3866018","identity":"rs-3866018","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}