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Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer
Cell Communication and Signaling volumeĀ 22, ArticleĀ number:Ā 45 (2024)
Abstract
Objectives
Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood.
Methods
We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53-/- cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved.
Results
We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (Pā=ā0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent.
Conclusions
Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer.
Introduction
Histological transformation to small cell lung cancer (SCLC), particularly from EGFR mutant lung adenocarcinoma (LUAD), has been recognized as a mechanism of EGFR tyrosine kinase inhibitors (EGFR TKIs) resistance as listed in the NCCN (National Comprehensive Cancer Network) clinical practice guidelines for non-small cell lung cancer (NSCLC) (https://www.nccn.org/, 2021.V3) [1, 2]. However, there still remain several critical questions about this phenomenon. One major topic is, case reports suggested that SCLC transformation can also occur after ALK inhibitor treatment [3, 4],PD1/PD-L1 immunotherapy [5], which implies that SCLC transformation is not restricted in EGFR mutant LUAD, while systematic real-world survey with a larger cohort is lacking.
Another issue that we are concerned about is the mechanism and the subsequent strategies of SCLC transition. RB1 and TP53 abnormalities are essentially universal in de-novo SCLCs [6] and also seem to be critical in transformed SCLC. Lee et al. [7]demonstrated that early alteration of RB1 and TP53 in primary LUAD indicated a high tendency of SCLC transition. Matthew et al. [8] verified that RB1 deletion is required for NSCLC-to-SCLC conversion because almost all of the transformed SCLCs exhibited homozygous deletion of the RB1 gene. Meanwhile, in Matthewās work, RB1 deletion was also observed in LUAD where TKI resistance occurred independently of SCLC transformation, suggesting that RB1 deletion alone is insufficient to explain the phenotypic switch [8]. Besides TP53 and RB1, thereās still lack of understanding of involved genes in SCLC transformation.
As reported, 3ā14% [2, 9] of EGFR-TKIs treated LUAD patients develop SCLC transformation and once transformation occurs, the treatment strategy is extremely limited coming with poor prognosis. LĆ©onie Ferrer et al. [10] reported a 10-month median overall survival (mOS) in 61 transformed cases. Nicolas Marcoux et al. [11] reported a 10.9-month mOS with frequent CNS metastases in 57 SCLC transformed cases. Almost all transformed cases were treated with platinum and etoposide (EP), which is the classic therapy of SCLC. Marcouxās study also reported that transformed SCLC showed unsatisfactory response to PD-1 antibodies. Thus, figuring out the mechanism and the involved key regulators of SCLC transformation is urgently demanded for new therapeutic drugs development.
Hence, in this real-world study, we investigated the occurrence of SCLC transformation in a relatively large NSCLC cohort. We also performed next-generation sequencing (NGS) on the paired tumor samples obtained from transformed cases to explore the potential genes contributing to the histological transformation.
Materials and methods
Patientsā clinicopathological characteristics
All participating NSCLC patients were pathologically diagnosed at Shanghai Pulmonary Hospital with key driver genes detection including EGFR, KRAS, and BRAF mutation and ALK, and ROS1 fusion by ARMS-PCR (AmoyDx, Xiamen, China) between January 2013 and December 2016. For all cases, clinical data (gender, age, clinical stage, smoking history and ECOG PS) were collected at the entry time. The pathological histology was determined with available surgical resected sample, biopsy or re-biopsy sample during the whole follow-up. EGFR-TKI therapies in this study included gefitinib and erlotinib. The short-term responses were evaluated after the first 2 cycles for chemotherapy, and after the first month for targeted therapy according to the Response Evaluation Criteria in Solid Tumors (RECIST). Progression-free survival (PFS) was defined as the period from the date of receiving treatment to that of objective disease progression or death or the last follow-up, and OS was defined as the period from the date of receiving treatment to that of death or the last follow-up.
Targeted tumor gene panel test using next generation sequencing
The paired tumor formalin-fixed paraffin-embedding (FFPE) specimens from resected tumor or biopsy were subjected to a tumor multigene NGS panel covering the whole exons and significant introns of 416 cancer-related genes (Nanjing Geneseeq Technology Inc., Jiangsu, China, CAP/CLIA-certified). Genomic DNA was extracted (QIAamp DNA FFPE Tissue Kit, QIAGEN) and quality controlled. The sequencing libraries were prepared with the optimized protocols according to the manufacturerās instructions (GeneseeqPrime, Jiangsu, China). The capture-enriched libraries were sequenced on Illumina HiSeq 4000 platform. Single nucleotide variation (SNVs) and indels were called by VarScan2 and Haplotype Caller/Unified Genotyper in GATK and filtered out by dbSNP database and data from 1000 Genome project. Gene fusions were identified by FACTERA and copy number variations (CNVs) were measured with ADTEx.
Cell culture
The human NSCLC cell line HCC827 was purchased from ATCC, and A549-TP53-/- was previously developed and preserved in the laboratory. Cells were cultured in DMEM (HyClone Lot. SH30585.02) supplemented with 10% FBS (Gibco Lot. 10,100,147) and 100U/ml penicillin/streptomycin (Gibco Lot. 15,140,122) at 37Ā Ā°C in 5% CO2 atmosphere.
Establishment of SMAD4 and RB1 knocking-out cell lines
SMAD4 and RB1 genes were knocked out by CRISPR-Cas9 technique (Genomeditech Shanghai, Co., LTD). SgRNA primers were designed on Zhang Fengās laboratory website: SMAD4, AGAGCAGGACAGCGGCCCGG and RB1, AGAGCAGGACAGCGGCCCGG. Monocolony screening was conducted to select the homozygotes-deleted clones after stable transfection lines were established. The cells were counted and diluted to 1 cell per 100ul of cell suspension and cultured at 100ul volume per well in 96-well cell plates. After regular incubation for 2 days, the cell clusters were observed under microscope and the single clones were transferred to 6 or 10Ā cm petri dish for further culture and proteins were extracted when cell amounts were huge.
MTT assay
For cell growth ability, cells were plated into 96-well plates at a density of 1000 cells per well. Culture medium were stripped off at different time points (24, 48,72, 96, 120, and 144Ā h) and cells were treated with 20Ā Āµl of MTT solution (5Ā mg/ml) at 37 ĖC for 4Ā h. Then 200Ā Āµl of DMSO was added to each well. The optical density (OD) was measured at 570-nm wavelength and each experiment was performed in triplicate. For drug sensitivity, cells were plated into 96-well plates at a density of 5000 cells per well and treated with drugs at different dilutions. Cells were cultured for 72Ā h and then culture medium were stripped off. MTT solution and DMSO and also OD measurement were then processed as described above.
Luciferase reporter gene assay and ChIP assay
The luciferase reporter plasmid under the control of ASCL1 promoter was constructed by Genomeditech, Shanghai, China. Briefly, the āā2000 to āā1 upstream area of the human ASCL1 gene was amplified by PCR using human genomic DNA and a set of forward and reverse primers containing custom MluI and XhoI restriction enzyme sites, respectively. The PCR product was cloned into to pGL3-Basic, which is a promoter-less luciferase vector. The PCR product was named as pGL3-Basic-H_ASCL1 promoter and the sequence was confirmed by sequencing analyses. The cells were transfected with pGL3-Basic-H_ASCL1 promoter plasmids, and after a transfection period of 24Ā h, the cells and lysates were collected. A Dual-Luciferase Reporter Assay System (Promega) was used to quantify luciferase activities following the manufacturerās instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. ChIP was performed following protocol of the ChIP assay kit purchased from Beyotime (Lot. P2078), and the antibodies used were as follows: SMAD4 (D3R4N) (CST #46,535) and MYC (Y69) (Abcam #ab32072). The PCR primer of ASCL1 was forward-AGCCATTTGTCCCTCCTGTG, reverse-CTCCTCTTACCTCTTCCTCCC.
Nude mouse tumor xenograft model
The BALB/C nude mice (Female, 6-week-old) were randomly divided into four groups (nā=ā4 per group) and cells (4āĆā106) were subcutaneously injected into the unilateral hind limbs of the nude mice. Tumor size was measured for 6 weeks after inoculation to calculating tumor volume using the equation (length Ć width2/2). Animals were killed 6 weeks after inoculation, and the tumors were then excised for further usage.
H&E staining, immunohistochemistry staining and Western blotting
Each resected tumor, biopsy or re-biopsy specimen from any primary tumor site, lymph node or metastases was examined with Hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining following regular protocols for histopathological observation. The histology for each specimen was determined independently by at least two pathology experts in the hospital. Cells were lysed with RIPA buffer containing a proteinase inhibitor mixture (Beyotime, P1005) and proteins were qualified by BCA assay (Beyotime, P0012s). Then western blot assay was performed following routine protocol. Antibodies used were listed in supplementary file.
Statistical analysis
All statistical analyses were conducted on SPSS platform (SPSS Inc., Chicago, IL, version 20.0), and the graphs were achieved on Graphpad (Prisim 9) platform. The differences of continuous variables were evaluated by ANOVAās test or t test. The relationships between classified variables were analyzed using Ļ2 test. A P valueā<ā0.05 at both sides was considered as statistically significant. The motif analysis was performed on ConTra v3 platform. The relevant parameters were set as follows: Reference organism: Homo sapiens, Genomic position: chr12:102955674ā102,957,673, and Stringency: coreā=ā0.95, similarity matrixā=ā0.85.
Results
SCLC transformation is irrespective of EGFR mutation status or histological subtype in NSCLC with poor prognosis and acquired neuroendocrine phenotype
One thousand four hundred seventy-four NSCLC patients were firstly diagnosed and 1074 with further treatment in our institution were further analyzed (Fig.Ā 1A; TableĀ 1). To our concern, 343 NSCLC patients received re-biopsy and 24 SCLC transformation cases were confirmed (24/343, 7.0%) including 13 EGFR mutant, 2 with positive ALK rearrangement and 9 wildtypes for either detected driver genes (TableĀ 2). In our study, the incidence rate of SCLC transformation was 5.65% (13/230) in EGFR mutant cohort and 9.73% (11/113) in EGFR wildtype cohort(Pā=ā0.16). The prevalence of SCLC transformation was 7.5% (21/279) in LUAD, and 4.7% (3/64) in LUSC, respectively (Pā=ā0.41). Our results revealed that SCLC transformation occurs regardless of EGFR mutation status or primary histological subtype (Pā>ā0.05, TableĀ 1). Two representative transformation cases were presented in Fig.Ā 1B and C.
The median time to relapse as transformed SCLC in our study was 23.0 months (95%CI, 19.4ā26.6) (Fig.Ā 1D). After transformation, the mOS was 9.0 months (95%CI, 7.5ā10.6), and the OS of EGFR wild type group was 4.0 months longer than that of EGFR mutant transformation cases (10.0 vs. 6.0 months, Pā=ā0.016) (Fig.Ā 1E). As typical cases presented above, transformed tumor tissues were positive for common NE markers such as Syn and CgA (Fig.Ā 1B and C). We also dynamically monitored serum NSE, a common NE marker, and as expected, we found a significant serum NSE increasement when SCLC transformation occurred as compared to initial NSCLC diagnosis (34.37āĀ±ā13.44 ng/ml vs. 16.03āĀ±ā3.01 ng/ml, Pā<ā0.0001) (Fig.Ā 1F). In a typical case (Fig.Ā 1G), serum NSE was always within the normal range at the initial diagnosis of LUAD and during the subsequent 5-month chemotherapy and 10-month Icotinib treatment, then a remarkable elevation was observed corresponding to the tumor progression on CT imaging when SCLC transformation was confirmed by re-biopsy. Furthermore, serum NSE decreased once SCLC therapy was administrated corresponding to a shrunk tumor on CT scan. A similar dynamic pattern of serum NSE level was also observed in the EGFR wildtype case who received surgery and multiline chemotherapies (Fig.Ā 1H).
Transformed SCLC harbored acquired gene alterations and SMAD4 was associated with neuroendocrine phenotype in NSCLC under TP53 inactivation
Among 24 transformation cases, only 8 cases had sufficient tumor tissues before and after transformation for further sequencing covering 416 cancer-related genes (Table S1). Totally, we identified 81 altered genes with 107 mutations (Table S2) and observed a high percentage of shared gene mutations in each case (Fig.Ā 2A). Itās widely known that inheriting EGFR mutations is an evidence of SCLC transformation, which was also identified in our cases (TableĀ 2). Besides, for SCLC transformation cases from EGFR wildtype LUADs, we identified the inheritance of RB1 and/or TP53 alterations in transformed SCLCs, such as TP53 R158L/E271X in Case 15, RB1 K294X in Case 19 (Table S3).
We also identified 41 genes which were mutated specifically in transformed SCLC tumors, and 18 genes showed a mutant rate of more than 25% in 8 sequenced cases such as RB1, TP53, NOTCH2, SMAD4, PIK3R2 and EPHA3 (Fig.Ā 2B). We analyzed the associations between these genes and NE related genes expression in NSCLC based on TCGA database, and in SMAD4-mutant LUADs, the ratio of ASCL1-upregulated LUADs was higher than that in SMAD4-wildtype cases (75.0% vs. 34.4%, Pā=ā0.027) especially under TP53 inactivation (Fig.Ā 2C; TableĀ 3). We also conducted pathway enrichment analysis and found that SMAD4 was involved in multiple pathways including cell size regulation and also neuron differentiation (Fig. S1B). Hence, we inferred that SMAD4 contributed to small-cell transition, especially by affecting the neuroendocrine phenotype under TP53 inactivation. We detected Smad4 expression in 16 small-cell transformed cases by IHC, and observed a significantly decrease of Smad4 expression in transformed SCLC specimen (IHC score: 2.5 vs. 1.7, Pā=ā0.027) (Fig.Ā 2F). In transformed cases, SMAD4 mutation was an inactivation alteration because no protein expression was detected in SMAD4 mutant SCLC specimen (Fig.Ā 2G).
SMAD4 deletion promoted tumor growth and induced multi-drug resistance in TP53 -inactivated NSCLC
To further explore the gene function, we knocked out SMAD4 in TP53-inactivated HCC827 and A549-TP53-/- cells and we further knocked out RB1 due to its essential role in SCLC formation. As shown in Fig. S2, we screened several clones labeled as HCC827-SMAD4-/-, HCC827-SMAD4-/-RB1-/-, A549-TP53-/--SMAD4-/- and A549-TP53-/--SMAD4-/-RB1-/- in which no Smad4 and/or Rb1 expression was detected.
We then explored the effect of Smad4 loss on cell growth. As a result, Smad4 deletion significantly promoted the HCC827 cell growth as compared to the control group, suggesting the tumor suppressor role of SMAD4. As expected, inactivation or co-inactivation of RB1 further enhanced the cell growth ability (Fig.Ā 3A). In in vivo experiments, we also observed the significantly increased tumor growth and tumor weight when SMAD4 was knocked out (Fig.Ā 3B, C and D), which was consistent with the results of in vitro experiment.
We treated EGFR-mutant HCC827 cells with Gefitinib, the 1st generation EGFR-TKI, and accordingly, the IC50 didnāt show significant changes when SMAD4 or RB1 was knocked out alone as compared to control group by MTT assay (57.08nM, 56.98nM vs. 56.76nM, Pā>ā0.05). However, the IC50 was significantly increased when SMAD4 and RB1 were co-inactivated as compared to control group (211.70 nM vs. 56.76nM, Pā>ā0.05). We also treated the cells with Pemetrexed, the most recommended regimen as first-line chemotherapy of LUAD. Consequently, Smad4 loss led to the significantly increased IC50 in HCC827 cells (SMAD4ā/ā, 19.5ĀµM vs. Control, 10.06ĀµM, Pā=ā0.0176). As compared to control group, the IC50 was significantly decreased when RB1 was knocked out alone (10.06ĀµM vs. 2.69 ĀµM, Pā=ā0.0003). However, if SMAD4 was co-inactivated, the sensitivity of cells to pemetrexed decreased further (SMAD4ā/āRB1ā/ā, 21.10ĀµM vs. Control, 10.06ĀµM, Pā=ā0.0057) (Fig.Ā 3E). Similar effect of Smad4 on cell growth was achieved in A549-TP53ā/ā cells while no obvious change of Pemetrexed sensitivity was observed (Fig.Ā 3F).
In addition, we detected the expression of PD-L1. Consequently, PD-L1 was down-expressed in SMAD4ā/ā cells while no obvious decrease was observed in RB1ā/ā cells as compared to control group by IHC staining and western blotting, which indicates the unsatisfactory treatment efficiency of PD-1/PD-L1 antibody therapy (Fig.Ā 3G).
SMAD4 loss induced acquired neuroendocrine phenotype independently of RB1 status by upregulating ASCL1 transcriptionĀ in vitroĀ andĀ in vivo
We detected NE markers in HCC827 cells and confirmed the positive expression of Syn and also ASCL1, the upstream regulator of multiple NE markers in SCLC when SMAD4 was knocked out. No obvious change of neuroendocrine makers expression was detected when RB1 was knocked out only (Fig.Ā 4A). Similar results were also observed in A549-TP53-/- cells (Fig. S3A). In murine xenografts, we found scattered CgA- and Syn-positive lesions in SMAD4-/- xenografts, and highly stained CgA, Syn and CD56 in SMAD4-/- RB1-/-xenografts by IHC staining (Fig.Ā 4B, IHC staining). SMAD4 is an important transcriptive factor (TF) regulating gene transcription. By motif analysis, we found Smad4 binding site (SBS) in the promoter region of ASCL1 gene, which has a base overlap with the E-box region, the binding site of MYC (Fig.Ā 4C). We further transfected established cell models with ASCL1 luciferase reporter vectors and we demonstrated the significant increasement of ASCL1 transcriptional activity in HCC827-SMAD4-/-RB1-/- cells (Pā=ā0.001, Fig.Ā 4D), while no obvious difference of activity was observed when RB1 was knocked out only. We further revealed the negative regulatory effect of SMAD4 on ASCL1 transcription by ChIP in SMAD4-wildtype NSCLC cells (Fig.Ā 4E). MYC binds gene promoters and activate transcription by forming a heterodimer with its primary partner MAX [12]. We also idenfied the interaction between SMAD4 and MAX in HCC827 cells by Co-IP (Fig.Ā 4F).
Therefore, we proposed the hypothesis that SMAD4 can compete with MYC for MAX to form transcriptional complexes to regulate ASCL1 transcription. In SMAD4-wildtype NSCLC, ASCL1 transcription is suppressed by MAX/SMAD4 complex, and MYC degradation is accelerated due to instability of protein without complex formation. When SMAD4 is loss-of-function mutant, ASCL1 transcription is activated by MYC/MAX complex, which will further regulate neuroendocrine phenotype in NSCLC (Fig.Ā 4H).
Myc inhibitor acts as the potential therapy for NSCLC with acquired neuroendocrine phenotype mediated by SMAD4 deficiency
As mentioned above, Myc was upregulated when SMAD4 was inactivated (Fig.Ā 4G). We treated each group with a pan-Myc inhibitor, and the result showed that the IC50 of HCC827-SMAD4ā/āRB1ā/ā was significantly decreased as compared to control group (4.08 ĀµM vs. 7.65 ĀµM, Pā=ā0.0075). Similar result was observed in A549-TP53ā/āSMAD4ā/āRB1ā/ā group although there was no statistical significance (2.74 ĀµM vs. 4.13 ĀµM, Pā=ā0.1776) (Fig.Ā 5A). Besides Myc inhibitor, Bcl2 and DLL-3 [13] antibodies are also potential targeted therapy targets for SCLC. We also detected the expression of Bcl2 and DLL-3 in established cell models. As a result, we found no obvious changes of Bcl2 and DLL-3 expression (Fig. S3E).
Chemotherapy still serves as the mainstream therapies for high-grade lung neuroendocrine tumors, which mainly refer to SCLC. We treated each group of cells with etoposide and irinotecan. As compared to control group, the IC50 of Etoposide was significantly decreased in HCC827-SMAD4ā/ā (14.0 ĀµM vs. 2.79 ĀµM, Pā<ā0.0001) and HCC827-SMAD4ā/āRB1ā/ā (14.0 ĀµM vs. 1.75 ĀµM, Pā<ā0.0001). Knockout of RB1 alone had no significant effect on Etoposide sensitivity as compared to control group (14.0 ĀµM vs. 13.4 ĀµM, Pā=ā0.8007) and similar results were observed in A549-TP53ā/ā group (Fig.Ā 5B). No obvious changes of irinotecan sensitivity were determined when SMAD4 and/or RB1 was deficient (Fig.Ā 5C).
Discussion
SCLC transformation is usually identified by re-biopsy at the time of relapse or disease progression [14,15,16].Recent reports [10, 11] demonstrated the rapid deterioration and shorter OS of transformed cases, which strongly requests urgent and more comprehensive understandings of this phenomenon. Here, our work indicates that SCLC transformation occurs regardless of EGFR mutation status/TKIs treatment and pathological type in NSCLC population, and also puts forward a set of potential genes contributing to the histological transformation.
To the best of our knowledge, this is the first time that an incidence of transformation in the EGFR wild type NSCLCs was reported. The majority of previous studies focused on EGFR mutant patients, only several individual cases reported the SCLC transformation from EGFR wild-type NSCLC [3, 17,18,19]. In our cohort consisting of 591 EGFR wild type patients, 11 transformed cases were identified in 113 individuals with repeat biopsy (11/113, 9.73%). The incidence rate in the EGFR mutant group is 5.65% (13/230), which is similar to previous study [20] involving 155 TKIs-resistant patients. Actually, in our work, the re-biopsy was recommended to some of the EGFR wild type patients by our clinicians based on their dynamic serum NSE change and disease progression, which may cause the data bias because of the intentional selection to avoid excessive medical examination. Moreover, this result also emphasizes the value of serum NSE monitoring in SCLC transformation as the changing curve shown in Fig.Ā 1G, H.
It is highly necessary to excavate the genetic predictors of pathological evolution towards SCLC. As previously studied [7, 21], the concurrent RB1 and TP53 alterations of initial LUAD tumors at the early-stage lead to a higher tendency to SCLC transformation. Correspondingly, our result also showed a higher mutant rate of RB1 and TP53 in funder LUAD tumors. Besides, we also found the inherited TP53 and/or RB1 mutations in transformed SCLCs from EGFR wildtype cases, which further supported the occurrence of SCLC transformation in EGFR wildtype cohort. By analyzing the specific altered genes in transformed SCLC tumors, we revealed a set of candidate genes, such as RB1, AKT1, SMAD4, and et al. Work by Park J.W. et al. [22] suggests a distinguished role of AKT1, besides TP53 and RB1, in small cell prostate cancer evolution. Previously, Smad4 was reported to be an independent factor of prognosis in NSCLC [23]. While the relationship between SMAD4 and NE phenotype is rarely reported in NSCLC. Nicky et al. [24] reported SMAD4 mutation in transformed SCLC patient while no further validation was conducted. Our work demonstrated that SMAD4 can promote the aggressive tumor behavior and induce NE phenotype and EGFR-TKI and pemetrexed resistance independently of RB1 status for the first time. Ascl1 is required for SCLC formation [25] ,our work also revealed that SMAD4 deficiency can upregulate ASCL1 transcription inducing NE phenotype in NSCLC. Consistent to our hypothesis [26], MYC amplification promoted tumor development with low Ascl1 expression though in SCLC mouse models.
For now, platinum-based etoposide still serves as the first choice for transformed SCLCs with nearly 50% [10, 11] clinical response rates. With accessible follow-up data in our study, the clinical response rate was 57.1% (8/14) in 14 transformed patients treated with platinum-etoposide. Itās a tough task to find target therapy for transformed SCLCs, as well as for primary SCLCs. In our study, we found the upregulated expression of Myc and an increased sensitivity to Myc inhibitor when SMAD4 was deficient. Moreover, the prognosis was significantly shortened for NSCLC patients with acquired NSE increasement during disease course, whatever SCLC transformation occurs or not (Fig. S2G). Thus, we inferred that SMAD4 deficiency led to drug resistance partially by inducing NE phenotype and Myc inhibitor could be the potential targeted therapy in NSCLC.
There are still several limitations and remaining questions in our work. Firstly, we just obtained 8 paired samples for sequencing. It will be more sufficient if more cases could be sequenced. Nevertheless, considering the low occurrence rate and the tiny repeat biopsy specimens, itās hard to receive abundant samples from transformation cases. Secondly, we just explored the function of SMAD4, and the contributions of other genes remain to be studied because it is highly possible that the transformation of SCLC requires the involvement of multiple genes.
Conclusions
To conclude, our results provide new insights into SCLC transformation from NSCLC, especially fill the gap in EGFR wide type cohort. SCLC transformation is a common phenomenon in NSCLC individuals, which is irrespective of EGFR mutation status, treatment regimens, and pathological subtype. In our study, we suggested that SMAD4 mutation can promote SCLC transformation and further lead to drug resistance in NSCLC. And the subsequent strategy is much more important from a clinical point of view. Our study proposed the potential therapeutic effect of Myc inhibitor besides traditional etoposide and iritecan. Some recent studies [27] also emphasized the potential of Myc inhibitors in SCLC treatment, which requires more study to support our view of Myc inhibitor for the treatment of transformed SCLC. Clinicians need to be highly aware of this phenomenon, and more in-depth researches are in pressing need.
Availability of data and materials
All data are available in the main text or the supplementary materials.
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Acknowledgements
We thank all the technicians (L. Tang, X.J. Yang, J. Zhang and A.X. Bi) from central lab for all the technical assistance in this study.
Funding
National Natural Science Foundation of China grant 82272897;Science and Technology Commission of Shanghai Municipality grant 19140901402, 17ZR1423500, 22dz1202500 and 201409003600; Health Commission of Shanghai Municipality grant 201940192; Youth talents subsidy Scheme of Shanghai Pulmonary Hospital grant fkcy1901.
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Conceptualization: J.Z., B.S., K.X.A. Methodology: X.D., M.X.S., D.L., J.X.C., K.X.Z., R.D.Z., L.P.Z. Investigation: J.X.C., K.X.Z., R.D.Z., L.P.Z., D.L., X.D. Visualization: X.D., M.X.S., B.S. Funding acquisition: B.S., J.Z., K.X.A., X.D. Project administration: K.X.A., B.S. Supervision: J.Z., B.S. Writing-original draft: X.D., M.X.S., D.L. Writing-review & editing: X.D., M.X.S., K.X.A.,B.S. All authors reviewed the manuscript.
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This study was conducted under approval of the appropriate Ethics Committees of Shanghai Pulmonary Hospital. All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All animal-involved experiments were carried out with the approval of Tongji University Animal Center.
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The authors declare no competing interests.
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Ding, X., Shi, Mx., Liu, D. et al. Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer. Cell Commun Signal 22, 45 (2024). https://doi.org/10.1186/s12964-023-01260-8
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DOI: https://doi.org/10.1186/s12964-023-01260-8