High frequency of alternative splicing variants of the oncogene Focal Adhesion Kinase in neuroendocrine tumors of the pancreas and breast

Abstract

The characteristic genetic abnormality of neuroendocrine neoplasms (NENs), a heterogeneous group of tumors found in various organs, remains to be identified. Here, based on the analysis of the splicing variants of an oncogene Focal Adhesion Kinase (FAK) in The Cancer Genome Atlas datasets that contain 9193 patients of 33 cancer subtypes, we found that Box 6/Box 7-containing FAK variants (FAK^6/7) were observed in 7 (87.5%) of 8 pancreatic neuroendocrine carcinomas and 20 (11.76%) of 170 pancreatic ductal adenocarcinomas (PDACs). We tested FAK variants in 157 tumor samples collected from Chinese patients with pancreatic tumors, and found that FAK^6/7 was positive in 34 (75.6%) of 45 pancreatic NENs, 19 (47.5%) of 40 pancreatic solid pseudopapillary neoplasms, and 2 (2.9%) of 69 PDACs. We further tested FAK splicing variants in breast neuroendocrine carcinoma (BrNECs), and found that FAK^6/7 was positive in 14 (93.3%) of 15 BrNECs but 0 in 23 non-NEC breast cancers. We explored the underlying mechanisms and found that a splicing factor serine/arginine repetitive matrix protein 4 (SRRM4) was overexpressed in FAK^6/7-positive pancreatic tumors and breast tumors, which promoted the formation of FAK^6/7 in cells. These results suggested that FAK^6/7 could be a biomarker of NENs and represent a potential therapeutic target for these orphan diseases.

Keywords

FAK ^6/7 / SRRM4 / neuroendocrine neoplasms / pancreas / breast

1 Introduction

Neuroendocrine neoplasms (NENs) are a heterogeneous group of rare (accounting for ~2% of all malignancies) but clinically important form of malignancies that occur in various organs, including gastrointestinal (GI) and respiratory tracts, central nervous system, and breast. NENs are divided into well-differentiated neuroendocrine tumors and poorly differentiated neuroendocrine carcinoma, which may have benign or aggressive behavior, respectively. Genetic abnormalities have been unveiled in some types of NENs [1,2], but alternative splicing profiles have rarely been investigated in NENs.

Tumors of pancreas comprise various subtypes such as pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine neoplasm (pNEN), pancreatic solid pseudopapillary neoplasm (SPN), acinar cell carcinoma (ACC), intraductal papillary mucinous neoplasm, mucinous cystic neoplasm (MCN), and squamous cell carcinoma (SCC) [3–6]. PDAC, which accounts for 90% of pancreatic tumors, is a recalcitrant malignant neoplasm that kills 466 000 patients each year worldwide, with a 5-year overall survival (OS) rate of no more than 10% [7,8]. pNEN accounts for 3%–5% and is the second most common type of pancreatic tumors [9], and it can be classified into differentiated pancreatic neuroendocrine tumor (PanNET) with a 5-year OS of 85.4% and poorly differentiated pancreatic neuroendocrine carcinoma (PanNEC) with a median OS of only 7.5 months [5,10]. SPN accounts for 2%–3% of pancreatic neoplasms and has a favorable prognosis [5,11–13]. The differentiation of tumor subtypes is critical for the treatment option for patients with different types of pancreatic neoplasms.

Patients with different subtypes of pancreatic tumors bear distinct molecular alterations. In PDAC, the most common driver mutations are mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), tumor suppressor TP53, cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), and SMAD family member 4 (SMAD4) [3,14,15]. Alterations in MEN1, alpha-thalassemia/mental retardation syndrome X-linked (ATRX), death-domain-associated protein (DAXX), and von Hippel-Lindau (VHL) are frequently found in PanNET [1], while mutations in TP53, KRAS, and retinoblastoma 1 (RB1) are common in PanNEC [15,16]. SPN harbors a high-frequency mutation in CTNNB1 (Catenin Beta 1) [17,18]. These genomic mutations pave the way of pancreatic carcinogenesis and provide potential targets for therapeutics. However, more genetic alterations that can help differentiate pancreatic tumor subtypes are still needed as guide for the selection of precise treatments for patients with different subtypes of pancreatic tumors.

Focal Adhesion Kinase (FAK), which is also known as protein tyrosine kinase 2 (PTK2), plays a vital role in cancer cell proliferation, metabolism, angiogenesis, tumor invasion, and metastasis [19,20]. In PDAC, FAK activity is significantly increased and is correlated with high levels of fibrosis and poor infiltration of CD8⁺ cytotoxic T cells [21]. FAK induces the secretion of interleukin-6 (IL-6) by cancer cells, which can act in concert with IL-4 produced by CD4⁺ T cells to upregulate the expression of programmed death ligand 2 (PD-L2) on tumor-associated macrophages, dendritic cells, and endothelial cells [22]. FAK activity is associated with poor survival of patients with PDAC, and the inhibition of FAK suppresses the growth and metastasis of pancreatic cancer cells [23,24], reduces metastasis after gemcitabine treatment, allows radiotherapy to prime tumor immunity, and unlocks responsiveness to immune checkpoint inhibitors [25–28]. FAK is overexpressed in PanNETs, and its inhibition induces apoptosis, inhibits PanNET proliferation, and synergizes with the mTOR inhibitor by preventing feedback AKT activation [24,29]. FAK inhibitor inhibits tumor growth, enhances the anti-tumor activity of cisplatin, and synergizes with vascular endothelial growth factor receptor-2 (VEGFR-2) blockade to reduce the metastasis of pancreatic neuroendocrine tumors in mice [30]. However, the mechanisms underlying FAK hyperactivation in pancreatic tumors remain to be elucidated.

FAK can be regulated by alternative splicing, a universal procedure through which exons and introns transform into mature mRNAs to generate multiple transcripts from a single gene [31]. For example, the wild-type (WT) FAK contains 32 exons, and FAK⁺, FAK⁶, FAK⁷, FAK^6,7, and FAK²⁸ are produced via exon inclusion manner, while FAK^Δ33, FAK^ΔE26, and FAK^ΔE5-27 are generated by exon skipping [32–37]. The FAK⁺ splicing variant includes an exon box that codes three amino acids proline, tryptophan, and arginine (PWR), and this variant is usually expressed in neurons [32]. FAK⁶, FAK⁷ and FAK²⁸ contain a coding box that encodes 6, 7, or 28 amino acids (Box 6, Box 7, Box 28), which are located after exon 14, 15, and 14 respectively, and are mainly expressed in brain. FAK^6,7 contains both Box 6 and Box 7 in the transcript [32,33]. The various splicing variants of FAK may play different roles in cancers [20]. FAK⁶ and FAK²⁸ are related to colorectal cancer metastasis [34], FAK^Δ33 is related to tumor aggressiveness in papillary thyroid carcinoma [36], and FAK⁶, FAK⁷, and FAK^6,7 are detected in Chinese non-small cell lung cancer (NSCLC) at a frequency of 4/91 (4.4%) and in The Cancer Genome Atlas (TCGA) NSCLC RNA-seq data at 79/1009 (7.8%). FAK^6,7 exhibits increased tyrosine kinase activity and promotes cell proliferation and migration in NSCLC [37]. However, the expression and roles of the splicing variants of FAK in pancreatic tumors have not been determined.

In the present study, we analyzed FAK splicing variants in the pan-cancer transcriptome data of TCGA that contain 9193 patients of 33 cancer subtypes, and found that FAK^6/7 were observed in 7 (87.5%) of 8 PanNECs. We tested FAK variants in 157 tumor samples collected from patients with different types of pancreatic tumors, and found that the incidence of FAK^6/7 reached 34 (75.6%) in 45 pNENs but was 2 (2.9%) in 69 PDACs. To verify the potential association between FAK splicing variants and NENs, we tested FAK^6/7 in breast neuroendocrine carcinoma (BrNECs) and found that these alternative splicing events (ASE) were seen in 14 (93.3%) of 15 BrNECs, but none of them was observed in 23 non-NEC breast cancers (BRCAs). We explored the pro-FAK alternative splicing mechanism and found that the expression of the splicing factor (SF) serine/arginine repetitive matrix protein 4 (SRRM4) was high in FAK^6/7-positive pancreatic tumors (PanTs) and breast tumors (BrTs), which promoted the formation of FAK^6/7 in cells. Therefore, FAK^6/7 may serve as a biomarker of NENs and may represent a therapeutic target for these orphan diseases.

2 Materials and methods

2.1 TCGA and the Cancer Cell Line Encyclopedia (CCLE) datasets

The TCGA level 3 IlluminaHiseq RNAseqV2 data were downloaded from the Broad GDAC Firehose [38]. The percent spliced-in (PSI) values of FAK were downloaded from the TCGA SpliceSeq PSI download page [39]. The positivity of FAK splicing variants are based on the two following criteria: PSI ≥ 0.05 and junction reads ≥ 2. Reads per kilobase per million mapped reads (RPKM) were used to measure the expression of the specific exon, and the log₂ (RPKM + 1) values were calculated. The PSI values of the FAK splicing variants of pancreas and breast cell lines were downloaded from the CCLE database, integrated to the Cancer Dependency Portal (DepMap), and then calculated [40]. The fragments per kilobase of transcript per million mapped reads (FPKM) and clinical data were downloaded, and then the FPKM data were transformed into transcript per million (TPM) data for differentially expressed gene (DEG) analysis between the WT FAK and FAK^6/7 groups by using Limma methods in the Sangerbox website [41]. The pan-cancer expression and survival analysis of SRRM4 were explored using the Timer and KM-plotter database [42–44].

2.2 Patient samples

The study was approved by the research ethics committees of all participating institutions; all samples were collected with written informed consent from the patient’s family. All tumor samples were analyzed, and the diagnosis and TNM stage were established by at least two independent expert pathologists. Tumor and adjacent normal tissues were immediately frozen in liquid nitrogen after surgical resection, and formalin-fixed paraffin-embedded (FFPE) tumor samples were collected and stored at 4 °C.

2.3 RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from tumor samples and cell lines using TRIzol reagent (Invitrogen, USA) or RNeasy FFPE kit for FFPE samples (Qiagen, Hilden, Germany) according to the manufacturer’s protocols and then reverse-transcribed into cDNA by using RevertAid reverse transcriptase (Thermo Scientific, Waltham, MA, USA). The cDNA was amplified using the HiScript III 1st Strand cDNA synthesis kit (Vazyme, Nanjing, Jiangsu, China). The primers F1/R1 were used to amplify the region from exon 14 to exon 16 in FAK cDNA, and subsequent primers F2/R2 were used for Sanger sequencing by BGI Genomics (Shenzhen, China). The sequences of primers are as follows [37]: F1: 5′- AAGCAAGGCATGCGGACACA-3′, R1: 5′-CTCTTTGAATCTCATAATCCCTGG-3′; and F2: 5′-TAACGGACAAGGGCTGCAAT-3′, R2: 5′-AAGTGTGCACAGCTCCATGA-3′.

2.4 Cell culture

Human PDAC cell lines AsPC-1, BxPC-3, CFPAC-1, HPAF-II, MIAPaca-2, PANC-1, SW1990, and embryonic kidney HEK293T (American Type Culture Collection, Manassas, VA, USA), two pNEN cell lines (BON-1 and QGP-1), and normal pancreatic epithelial cell line HPDE6-C7 (Cell Resource Center, Chinese Academy of Medical Sciences, Beijing, China) were grown in Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640, or DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco/BRL, Grand Island, NY) at 37 °C with 5% CO₂. Total RNA was extracted and reverse-transcribed into cDNA, then used to amplify the region from exon 14 to exon 16 in FAK cDNA, and finally sequenced by BGI Genomics (Shenzhen, China) with Sanger sequencing platform.

2.5 Transient overexpression and Western blot analysis

The Flag- and HA-tagged plasmids were constructed and confirmed by Sanger sequencing. The 293T cells were transiently transfected with indicated plasmids by using Lipofectamine 3000 reagent (Invitrogen, Frederick, MD). After 72 h, the RNA was extracted as described above, and the proteins were harvested from cells by using the RIPA lysis buffer. Western blot analysis was carried out to confirm transfection. Proteins were subjected to immunoblot by using the indicated antibodies, including anti-Flag M2 (1:1000, Sigma, St. Louis, MO), anti-β-actin, and anti-GAPDH (1:10 000, Proteintech, Wuhan, China).

2.6 FAK minigene splicing assay

For the minigene splicing assay, the minigene (MG) construct was designed to express mRNA from FAK exon 14, 15, 16, Box 6, and Box 7 along with 250 base pairs (bp) of intronic nucleotides flanking Box 6/7 and 300 bp flanking exon 14, 15, and 16. The sequence was cloned into the pcDNA3.1(+) empty vector for FAK⁶, FAK⁷, and FAK^6,7 minigenes (FAK⁶-MG, FAK⁷-MG, and FAK^6,7-MG). The UGC motif in the upstream of Box 7 near poly-pyrimidine tract was mutated to UAC for the synthesis of the minigene mutant (FAK⁷-MUT). Minigene vector was co-transfected with HA-SRRM4 in 293T cells, RNA was harvested 24 h post-transfection, and FAK splicing variants was detected as described above. The primers F1/R3 and F3/R1 used to amplify FAK⁶ and FAK⁷ splicing variants are as follows: F3: 5′-AAACAGATGATTATGCTGAGATTATAGAT-3′; and R3: 5′-TTGAGGGCATGGTGTAAGTATCTT-3′.

2.7 Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) and SPSS 26 (IBM, Armonk, NY, USA). The significance of qualitative variables was compared using two-sided Chi-square test or Fisher’s exact test. Differences between two groups were analyzed using two-sided Student’s t test. Survival analyses were performed using the demographic characteristics of patients of our cohorts and the Online Survival Analysis Software and log-rank test. P values of less than 0.05 were considered statistically significant.

3 Results

3.1 FAK splicing variants in TCGA data sets

The TCGA database, which contains 9193 cases of 33 cancer types, was used to evaluate FAK RNA expression, from which FAK splicing variants were found in tumor samples. The ratio of Box 6 and/or Box 7 (Box 6/7)-containing FAK variants (including FAK⁶, FAK⁷, and FAK^6,7) quite differed across different cancer types (Fig.1). The ratios of FAK^6/7 variants were high in tumors in the central nervous system, with values of 92.4% in low-grade glioma (LGG), 83.8% in pheochromocytoma and paraganglioma (PCPG), and 54.9% in glioblastoma multiforme (GBM). The ratios of FAK^6/7 variants were 26.7% in ovarian (OV) cancer, 17.5% in uterine carcinosarcoma (UCS), 8.3% in lung adenocarcinoma (LUAD), and 7.4% in lung squamous cell carcinoma (LUSC) (Fig.1). The ratios of FAK^6/7 variants were low in lymphoid neoplasm diffuse large B cell lymphoma (DLBC), acute myeloid leukemia (AML), and thymoma (THYM) (Fig.1).

FAK^6/7 splicing variants were identified in 27 (15.17%) of 178 patients with PanTs (Fig.1), whereas other forms of splicing alternatives were not detected. In TCGA, the ratio of FAK^6/7 reached 7 (87.5%) in 8 PanNECs and was 20 (11.76%) in 170 PDACs (Fig.1). In PanTs, the PSI values were 0–0.89 and 0–0.93 for Box 6 and Box 7, respectively (Fig.1). By analyzing the corresponding splice junctions and PSI values, 27 (15.17%) PanTs were identified as “Box 6 and/or Box 7 (Box 6/7) positive”, and 15 (8.43%) patients owned both Box 6 and Box 7 (Box 6,7; Fig.1). The PSI values did not substantially differ in smokers compared with non-smokers (Fig.1). The median OS of FAK^6/7+ PDACs was 517 days, which is slightly but not significantly shorter than that of FAK^6/7− patients (Fig.1), possibly because of the small sample size of the FAK^6/7+ patients.

3.2 Pancreatic tumors of Chinese ancestry

The above results suggest that FAK^6/7 variants are associated with PanNECs. We therefore collected tumor samples from 157 patients with different types of pancreatic cancers (Tab.1) to analyze the relationship between FAK splicing alternatives and pNENs. These samples included 85 tumors and 21 normal tissues, which were immediately frozen in liquid nitrogen after surgical resection, and 72 FFPE tumor samples. Among the 157 patients, 70 (44.6%) were males, 86 (54.8%) were females, and 131 (83.4%) were below the age of 65 years. These participants included 45 (28.7%) patients with pNEN, 40 (25.5%) patients with SPN, and 69 (43.9%) patients with PDAC (Tab.1). These patients had variable clinical outcome, that is, some patients with PDAC had a short OS, whereas patients with PanNET and SPN had a relatively favorable prognosis (Fig.2). The incidence of FAK^6/7 variants differed among PanT subtypes, with values ranging from 2.9% in PDAC to 81.08% in PanNET (Fig.2).

3.3 High frequency of FAK splicing variants in pNENs of Chinese ancestry

Among the 45 patients with pNEN, 26 (57.8%) were males, 19 (42.2%) were females, 37 (82.2%) were below the age of 65, 8 (17.8%) were 65-years and over, 8 (17.8%) were smokers, 35 (77.8%) were non-smokers, 37 (82.2%) were diagnosed as PanNET, and 8 (17.8%) were diagnosed as PanNEC (Table S1). Based on the RT-PCR results, seven normal pancreatic tissues expressed WT FAK (Fig.3). Among the 45 patients, 34 (75.6%) cases bore FAK splicing variants, including 6 FAK⁶/FAK⁷ and 28 FAK^6,7 (Table S1). Among the 37 patients with PanNET, 30 (81.1%) had FAK splicing variants, including 5 FAK⁶/FAK⁷ and 25 FAK^6,7 (Fig.3). Sanger sequencing of the PCR products confirmed these findings (Fig.3). Among the eight patients with PanNEC, four (50%) had FAK splicing variants including 1 FAK⁶/FAK⁷ and 3 FAK^6,7. No association was found between FAK^6/7 expression and gender, age, smoking status, and TNM stage (Table S1). FAK^6/7 splicing variants were not associated with the overall survival (OS) of patients with pNEN and PanNET (Fig.3 and 3D). Although the P value was not significant because of the small sample size, FAK^6/7 indicated a potential association with poor prognosis in PanNECs (Fig.3).

3.4 High frequency of FAK splicing variants in SPN

Among the 40 patients with SPN, 33 (82.5%) were females, 7 (17.5%) were males, 39 (97.5%) were younger than 65 years, 38 (95.0%) were non-smokers, and 35 (87.5%) were at stage I–II (Table S2). The expression of FAK splicing variants in SPN was detected by RT-PCR, and the results showed that the six normal tissues expressed WT FAK, and 19 (47.5%) of the 40 patients harbored FAK splicing variants, including 16 FAK⁶/FAK⁷ and 3 FAK^6,7 (Fig.4), as confirmed by Sanger sequencing of the PCR products (Fig.4). FAK splicing variants were not associated with gender, age, smoking status, or TNM stage (Table S2). In addition, FAK splicing variants were not associated with OS of the patients (Fig.4).

3.5 Low frequency of FAK splicing variants in PDACs

FAK splicing variants were tested in tumor samples harvested from 69 Chinese patients with PDAC. The participants included 36 (52.2%) males and 32 (46.4%) females, 52 (75.4%) were younger than 65 years, and 17 (24.6%) were 65 years and over (Table S3). While the eight normal tissues expressed WT FAK, only two (2.9%) PDAC cases harbored FAK splicing variants, including one FAK^6,7 and one FAK⁶/FAK⁷ (Fig.5). Sanger sequencing confirmed the presence of these variants in the patients (Fig.5). FAK^6/7 variants were also detected in one of two ACCs and one mucinous cystic neoplasm (MCN; Fig.5 and 5B).

3.6 FAK^6/7 in breast neuroendocrine tumors

The above results suggest a potential association between FAK splicing variants and neuroendocrine tumors. This possibility was tested by testing the expression of FAK^6/7 in breast neuroendocrine carcinomas (BrNECs). In 1092 patients with BrTs, the most commonly diagnosed malignant tumor worldwide [45], of TCGA datasets, the PSI values were 0 to 0.67 and 0 to 0.96 for Box 6 and Box 7, respectively (Fig.6). FAK^6/7 splicing variants were identified in 147 (13.46%) of the patients, i.e., 1 in 1 BrNEC, 1 in 1 breast mixed cancer (BrMC) and 145 (13.3%) in 1090 breast non-neuroendocrine carcinoma (BrnonNEC), and 46 (4.21%) patients harbored both Box 6 and Box 7 (Fig.6). The median OS of FAK^6/7+ BrTs was 115 months, slightly but not significantly shorter than that of FAK^6/7− patients (Fig.6).

To test the expression of FAK^6/7 in our own BrT cohort, tumors were collected from 42 FFPE BrT samples, including 15 (35.7%) BrNECs, 23 (54.8%) BrnonNECs, and 4 (9.5%) BrMCs. Among these patients, 41 (97.6%) were females, 34 (81.0%) were below the age of 65, and 25 (59.5%) were non-smokers (Tab.2). Based on the RT-PCR assay, FAK^6/7 was positive in none of the 23 BrnonNECs, 4 (100%) of 4 BrMCs, and 14 (93.3%) of 15 BrNECs (Fig.6 and 6E). These findings were confirmed by Sanger sequencing of the PCR products (Fig.6).

The FAK^6/7 expression in pancreatic and breast cell lines was analyzed based on CCLE data on the Cancer Dependency Portal (DepMap) by determining the PSI values of FAK and the positivity of Box 6/Box 7-containing splicing variants. Among the 41 pancreatic cell lines tested, 1 line (2.4%; QGP1 that is a pNEN cell line) harbored FAK^6/7 transcripts (Fig.7), as confirmed by RT-PCR (Fig.7). Among the 51 breast cell lines tested, 6 (11.76%) harbored Box 6/Box 7 in FAK transcripts (Fig.7).

3.7 SRRM4 promotes FAK^6/7 formation

The splicing regulators that may promote the formation of Box 6/Box 7 splicing events were investigated by analyzing the DEGs between patients with FAK^6/7 and WT FAK in TCGA PanT and BrT data sets and screening for potential SFs and RNA binding proteins (RBPs) [46,47]. Interestingly, six candidate SFs, i.e., SRRM4, neuro-oncological ventral antigen 1 (NOVA1), DnaJ Heat Shock Protein Family (Hsp40) Member C6 (DNAJC6), CUGBP Elav-like family member 3 (CELF3), CUGBP Elav-like family member 4 (CELF4), and ELAV-like RNA binding protein 3 (ELAVL3), were overexpressed in FAK^6/7-bearing PanTs and BrTs (Fig.8).

SRRM4 is an SF that can regulate microexons, which comprise a class of exons that are 3–27 nucleotides in length. SRRM4 is highly expressed in neurons and plays a crucial role in brain development and neuronal differentiation [47,48]. NOVA1 represents an evolutionarily conserved SF that plays a key role in synapse formation and neuron development [49,50]. The role of these two SFs in FAK^6/7 formation was determined, and the results show that the exogenous expression of SRRM4 induced the formation of FAK^6/7 in 293T cells (Fig.8 and 8C). However, exogenous expression of NOVA1 in 293T cells did not result in FAK^6/7production (Fig.8).

SRRM4 binds to UGC motif in the upstream of the microexons of pre-mRNA and regulates its splicing via the C terminus enhancer of microexon (eMIC) domain [48,51]. We constructed an inactive SRRM4 deletion mutant (DM) by deleting its eMIC domain (Fig.8), and found that while SRRM4 was able to produce FAK splicing alternatives in 293T cells, SRRM4-DM failed to do so (Fig.8). We constructed three WT MG vectors containing Box 6 and/or Box 7 with adjacent exons named FAK⁶-MG, FAK⁷-MG, and FAK^6,7-MG, and a mutant FAK⁷-MG vector (FAK⁷-MUT), in which the nucleotide G was substituted by A in UGC motif (TGC in DNA, Fig.8). We found that co-expression of FAK-MG and SRRM4 promoted Box 6 and/or Box 7 expression in 293T cells, whereas FAK⁷-MUT did not induce FAK splicing variants (Fig.8).

3.8 SRRM4 in cancers

The expression of SRRM4 in TCGA pan-cancer data was further investigated, and the results indicate that this SF was highly expressed in LGG, GBM, and PCPG (Fig.8), which showed high expression levels of FAK^6/7 (Fig.1). SRRM4 expression was high in BRCA, LUAD, and LUSC tumor tissues compared with their counterpart normal tissues (Fig.8). The correlations between SRRM4 and FAK splicing in pancreatic and breast cancers were analyzed using the transcriptome data of TCGA. The results showed that in pancreatic cancers, Box 6/7-containing FAK splicing variants (expressed as PSI values) were associated with SRRM4 expression levels (Fig.8 and 8J), whereas in breast cancer, the association was relatively weak (Fig.8 and 8L). In LGG (Fig. S1), GBM (Fig. S2), and PCPG (Fig. S3), Box 6/7-containing FAK splicing variants were also associated with SRRM4 expression levels. We analyzed the potential association between SRRM4 expression level and survival time of the patients, and found that high SRRM4 expression was associated with favorable OS and relapse-free survival (RFS) of PanT patients (Fig.8 and 8N). However, SRRM4 expression level was inversely associated OS and RFS in BrT patients (Fig.8 and 8P).

4 Discussion

FAK plays a crucial role in normal pancreatic function and cancer development. It regulates pancreatic branching morphogenesis [52] and controls the onset of pancreatic cell differentiation [53]. Nuclear FAK controls chemokine CCL5 transcription, regulatory T cells, and evasion of anti-tumor immunity [54]. In comparison with the FAK activity in fibroblasts from healthy pancreas, that in cancer-associated fibroblasts (CAFs) is markedly increased [25], which regulates malignant cell metabolism [55] and is inversely associated with the OS of PDAC patients [25]. FAK in endothelial cells acts as a regulator of tumor chemosensitivity [56], and its suppression reduces PDAC metastasis after gemcitabine treatment [26,56]. FAK inhibition suppresses the growth and metastasis of pancreatic cancer cells concomitant with altering the TME [23,24]. In BrTs, FAK promotes tumor proliferation and metastasis and is related to tumor angiogenesis and vascular permeability in TME [57]. In addition, FAK sustains a pool of breast cancer stem cells with protumorigenic functions [58]. FAK in mononuclear phagocytes in TME recruits natural killer (NK) cells and contributes to the immune escape and increased tumor size [59]. FAK suppression confers PDAC responsive to immune checkpoint inhibitor [21], inhibits fibrosis and reduces immunosuppressive cells within primary tumors, suppresses M2 macrophages, and markedly decreases tumor spread [25]. FAK inhibition-induced stromal reprogramming overcomes radiation resistance to allow for immune priming and response to checkpoint blockade [27]. FAK inhibition synergizes with mTOR inhibitor for PanNETs [24]. Therefore, FAK is critical to carcinogenesis, therapeutics, and drug resistance in pancreatic and BrTs, and the mechanisms of FAK hyperactivation warrants further investigation.

The alternative splicing of pre-mRNA is a universal procedure for the transformation of exons and introns into mature mRNAs in eukaryotes. More than 95% of human protein coding genes are alternatively spliced into different mRNA variants, and some of them can be translated into proteins, demonstrating the important roles of alternative splicing in gene expression regulation and proteome diversity [60]. Alternative splicing plays critical roles in diverse biological processing and is frequently dysregulated in diseases, including cancers [61]. The alternative splicing variants of FAK have elevated kinase activity and can promote proliferation and metastasis in breast cancer, papillary thyroid carcinoma, colorectal cancer, and NSCLC [34–37]. Here, we reported for the first time that FAK splicing variants FAK^6/7 were expressed at different ratios in different types of PanTs, at frequencies of 75.6% in pNEN, 47.5% in SPN, and 2.9% in PDACs, suggesting that FAK splicing variants could be a biomarker of NENs. This possibility was confirmed in BrTs, in which FAK^6/7 was positive in 93.3% of samples of BrNEC, 100% of samples of BrMCs, and none of the samples of BrnonNEC, confirming the association between FAK^6/7 and NENs. Considering that FAK promotes cancer stem cells [58], CAFs [25], and immunosuppressive cells and facilitates tumor proliferation, metastasis, chemoresistance, angiogenesis [57], and immune escape, FAK^6/7 with elevated tyrosine kinase activity [37] may further enhance their tumorigenic effects in NENs. Moreover, different types of tumors originated from a same organ may have different splicing variants of the same oncogene, and different mechanisms for the generation of these splicing alternatives warrant further investigation.

The prognosis of pancreatic neoplasms varies with histopathologic subtypes. Generally, PDAC has the worst prognosis among the pancreatic cancers because of its unresectability and metastasis status and resistance to current therapies [3]. A recent study provides a detailed prognosis information of Caucasian/African-American PanTs, in which the 5-year survival rates of 84% and 97% were obtained for pNENs and SPNs, respectively [6]. In the present study, Chinese pNENs and SPNs also had a relatively favorable prognosis. The results indicate that the frequency of FAK^6/7 was much higher in pNENs and SPNs than in PDACs in both the TCGA-PanTs and our cohorts. Therefore, FAK^6/7 may help in the differentiation of different types of pancreatic tumors and represent a potential prognosis marker. Furthermore, the incidence of FAK^6/7 is among the highest in cancers of central nervous system, including LGG, PCPG, and GBM. Among the 43 pancreatic tumor cell lines, FAK^6,7 was only detected in a pNEN cell line QGP1. In BrTs, FAK^6/7 is highly expressed in BrNECs and BrMCs but not in BrnonNEC. Collectively, these findings suggest that FAK^6/7 might be related to neuroendocrine function and can be used as a universal marker to help in differentiating the diagnosis of neuroendocrine and non-neuroendocrine tumors. These possibilities warrant further investigation.

SRRM4, a neural-tissue specific SF that is predominantly expressed in brain, plays a crucial role in neurogenesis, neuronal maturation, differentiation, and function [62]. Aberrant SRRM4 expression is involved in autism spectrum disorder and cancers [63,64]. SRRM4 drives the neuroendocrine transdifferentiation of prostate adenocarcinoma to neuroendocrine cancer under anti-cancer therapy by mediating its target genes’ alternative splicing including RE1 silencing transcription factor (REST), Bif-1 and BHC80 [65-68]. REST is a transcriptional repressor of most neuronal genes that acts by binding to target genes through its zinc finger domains [69]. SRRM4 promotes the expression of splicing variants sREST, which lacks zinc finger domains, leading to neuroendocrine phenotype in small cell lung cancer and prostate neuroendocrine cancer [66,70]. In the present study, we found that FAK is a target gene of SRRM4, which promotes FAK^6/7 splicing variants in the cellular model. SRRM4 expression was associated with the favorable prognosis of PanTs possibly because of its role in the production of FAK^6/7, which is associated with pNENs. In BrTs, FAK^6/7 expression levels were not associated with prognosis, while SRRM4 expression was associated with poor prognosis. Therefore, SRRM4 may play different and complicated roles in the disease pathogenesis of PanTs and BrTs, and this difference warrants further investigation. Together, these results provide guidance for the understanding of the roles of FAK^6/7 and SRRM4 in the tumorigenesis of neuroendocrine neoplasms and targeted therapeutics for these orphan diseases.

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