DOI: 10.31038/CST.2023824

Simple Summary

The nucleotide excision repair (NER) pathway involves more than thirty protein-protein interactions and removes DNA adducts caused by chemotherapy drugs. The key genes of NER are often over-expressed in cancer cells and alterations of this pathway are responsible for increased or decreased sensitivity to specific therapeutic agents. This is particularly relevant in soft tissue sarcomas (STS), rare mesenchymal-originated tumors whose underlying mechanisms still lack understanding. Altogether the NER pathway components can be potential therapeutic targets in STS. The subtle regulation of NER activity may be clinically relevant as a surrogate prognostic marker or to predict sensitivity to chemotherapy agents. Further prospective evaluation of NER should be performed to address this question.

Abstract

Soft tissue sarcomas (STS) are low-incidence, mesenchymal-derived tumors represented by more than 50 his to types. Despite the latest developments, the rates of patients developing recurrent and metastatic disease are high. Many of the mechanisms underlying STS are still unknown, but there is evidence of the possible role of DNA damage response (DDR) pathways. DDR pathways include a variety of pathways used by cells to repair DNA damage of various kinds; they also have roles in protecting cancer cells from exogenous agents that target DNA, for these reasons are one of the main targets of potential anticancer therapeutic strategies. Nucleotide excision repair (NER) is one of the key repair pathways that can remove various bulky DNA lesions, often given by UV light, and is the main repair mechanism of DNA damage caused by carcinogens and chemotherapeutic drugs. Defects in NER are often the cause of several autosomal recessive genetic diseases. Variations in NER pathway actors can lead to a NER proficiency or NER deficiency condition, and this can be a risk, prognostic, and treatment response factor in cancer. This review focuses on the association between variations in the NER pathway in STS and is intended to point to NER as a pathway to focus on in the next future to optimize the treatments in use and improve the possibilities of personalizing therapies in STS patients in clinical practice.

Keywords

DNA damage response (DDR) mechanism, Nucleotide excision repair (NER), Soft tissue sarcoma (STS), Chemosensitivity

Introduction

Soft tissue sarcomas (STS) are rare tumors with more than 100 different histological subtypes. The scientific community has focused over the years on the search for biomarkers in STS [1] and the identification of variations at the genomic [2], expression [3], and protein [4] level.

Maintaining the integrity of genetic material is critical for the survival of all cell lines, yet various factors, both endogenous and exogenous, can compromise DNA stability. DNA damage repair (DDR) mechanisms are necessary for the maintenance of genome integrity. This is particularly important in cancer cells, in which mechanisms of resistance and sensitivity to radio- and chemotherapeutic cytotoxic agents are directly controlled by DDR pathways. For these reasons, DDR pathways are one of the main targets of potential anticancer therapeutic strategies [5]. The main DDR pathways are direct repair (DR), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), non-homologous end joining (NHEJ), and homologous recombination repair (HRR) [6]. The absence or deficiency of a specific DDR mechanism can result in genomic instability and tumor progression. Certain modifications of specific genes of a DDR pathway are typical, with some frequency, of some specific cancers [7]. Transcriptomic profiling of tumor tissues suggested codependences between DDR pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. Somatic alterations in the NER pathway, especially in ERCC genes, are common in various types of cancer. In a 2007 study, Castro et al showed that in cancer cells, NER and apoptosis pathways are the most impaired, with a high diversity of gene expression profiles in comparison to normal cells [8]. Other studies have shown how a deficiency in the NER pathway correlates with increased sensitivity to irofulven and cisplatin [9,10] and decreased sensitivity to trabectedin [11]. Moreover, inhibiting NER has been shown to increase sensitivity to alkylating agents in multiple myeloma cases [12]. DDR pathway alterations are present in numerous histologic subtypes of sarcoma. In a study conducted on STS specimens, at least one pathogenic mutation of the DDR pathway was detected in 15.9% of the patients, the most altered gene was ATRX (10%) and furthermore mutations were observed in 25 sarcoma subtypes. [13]. Recent studies have analyzed the genotype and expression profile of NER genes in STS patients, showing a correlation between ERCC1 and ERCC2 specific single nucleotide polymorphisms (SNPs) and a higher expression of both genes [14].

Nucleotide Excision Repair (NER) Pathway

NER mechanism recognizes and repairs various types of DNA damage caused by UV irradiation, cisplatin, and other damaging agents. NER pathways can be classified as either global genome repair (GGR), which repairs DNA damage anywhere in the genome, or transcription-coupled repair (TCR), which specifically restores DNA strands that are being transcribed (Figure 1). NER mechanisms rely on a series of reactions: recognition of DNA damage, unwinding double-strand DNA in the neighborhood of the damage, excision of the damaged nucleotides, and filling of the single-stranded gap by DNA synthesis. In GGR, DNA damage is recognized by XPC/Rad23 (xeroderma pigmentosum, C/Rad23 complementation group) or UV-DDB (UV-damaged DNA binding protein) [15] while in TC-NER, DNA damage blocks RNA polymerase II (RNAPII) interacting with CSB (ERCC excision repair 6, chromatin remodeling factor) and CSA (ERCC excision repair 8, subunit of CSA ubiquitin ligase complex)-CSB. After damage recognition, in both pathways, RNA polymerase II H transcription initiation factor (TFIIH) is recruited. It is subsequently recruited to the XPG (ERCC excision repair 5, endonuclease) complex, a single-stranded DNA-specific endonuclease. TFIIH unwinds DNA in the vicinity of damage, XPD (ERCC excision repair 2, helicase subunit of the TFIIH core complex), XPB (ERCC excision repair 3, helicase subunit of the TFIIH core complex) and XPA (DNA damage recognition and repair factor) are in charge of recognizing and verifying the damage. XPA binds to the chemically altered nucleotides in a single strand of DNA and recruits the XPF (ERCC excision repair 4, endonuclease catalytic subunit)-ERCC1 (ERCC excision repair 1, endonuclease non-catalytic subunit) catalytic subunit, which makes a cut on the damaged strand of 5′ to extract the damage. Next, XPG makes a 3′ cut that leads to the excision of a single-stranded DNA fragment containing the damage. Then thanks to PCNA (proliferating cell nuclear antigen) and DNA polymerase δ or ε new DNA is synthesized, finally DNA ligase 1 or 3 seals the DNA [16]. Defects in the NER pathway can be attributed to several inherited human diseases, including xeroderma pigmentosum (XP), an autosomal recessive genetic disease characterized by increased sensitivity to UV radiation [17] (Figure 1).

Figure 1: NER proficient and NER deficient tumoral cells. Effects of variations in the NER pathway leading to a NER proficient condition (left) with correct repair of DNA damage or NER deficient (right) with DNA damage persisting. The NER deficient condition can be reversible, resulting in DNA repair, or irreversible, resulting in permanent DNA damage that can lead to cellular damage.

ERCC1

The product of this gene is required for the repair of DNA lesions such as those induced by UV light or formed by electrophilic compounds including cisplatin. The encoded protein forms a heterodimer with the XPF endonuclease, and the heterodimeric endonuclease catalyzes the 5′ incision in the process of excising the DNA lesion. The heterodimeric endonuclease is also involved in HRR and in the repair of inter-strand crosslinks [18]. Overexpression of ERCC1 is correlated with better progression-free survival (PFS) in patients treated with doxorubicin plus trabectedin [19] and favorable overall survival (OS) [19]. Additionally, high expression of ERCC1, and BRCA1 haplotype were associated with the improved progression-free rate (PFR), PFS, and OS in STS [20]. Increased ERCC1 and XPF expression were associated with improved disease-free survival (DFS) and distant disease-free survival (DDFS) in STS [21]. A study carried out on STS patients showed that regarding the SNP rs11615, the alternative allele has a higher germline frequency than the general population and ERCC1 is overexpressed in 75% of STS samples analyzed compared to healthy corresponding tissue and its expression varies according to the genotype [14].

ERCC2 (XPD)

ERCC2 is part of the BTF2/TFIIH complex, which is essential in TCR. The translated protein has ATP-dependent DNA helicase activity and belongs to the RAD3/XPD subfamily of helicases. Defects in this gene are related to Cockayne syndrome, XP cancer-prone complementation group D syndrome, and trichothiodystrophy. [22]. ERCC2 gene is overexpressed in STS and its expression varies according to the genotyping of rs13181 and rs1799793, in addition, these SNPs have a higher frequency than the general population [14]. Additionally, ERCC2 is mutated in 3% of epithelioid sarcoma and 6.5% in perivascular epithelioid cell tumors and is significantly associated with increased homologous recombination deficiency (HRD) scores [13].

ERCC3 (XPB)

This gene encodes an ATP-dependent DNA helicase that is a subunit of basal transcription factor 2 (TFIIH) and, therefore, also functions in class II transcription. Mutations in this gene are associated with XP B, Cockayne’s syndrome, and trichothiodystrophy [23] ERCC3 overexpression is associated with disease progression in STS patients treated with trabectedin [24]. A study analyzing the genetic background, by whole-exome analysis, of a family with a 4-year-old child who has a Li-Fraumeni tumor (often associated with STS) hypothesized that ERCC3 may be a potential TP53-related modifier candidate responsible for accelerated tumor onset by the proband compared with the mother, who carries the same TP53 mutation [25].

ERCC4 (XPF)

XPF forms a complex with ERCC1 by playing a role in the 5′ incision made during NER. This complex is a DNA repair-specific endonuclease that interacts with meiotic structure-specific essential endonuclease 1 (EME1). Variations in this gene can underlie xeroderma pigmentosum complementation group F (XP-F), or xeroderma pigmentosum VI (XP6) [26]. In a study that performed targeted genomic sequencing within an Asian cohort of sarcoma patients, a truncating mutation in ERCC4 (p.Cys723*) was found in two patients with sarcoma diagnosed under 25 years of age [27]. In a retrospective study on angiosarcoma, ERCC4 was found mutated in 6% of patients [28].

ERCC5 (XPG)

This gene encodes a single-stranded DNA-specific endonuclease that makes the 3′ incision in DNA excision repair. XPG also plays a role in RNAPII transcription. Variations in this gene can cause xeroderma pigmentosum complementation group G (XP-G) and Cockayne syndrome [29]. A study of 113 STS samples showed a correlation between high expression of the common allele (aspartic acid at codon 1104) and better PFR, PFS, and OS [20]. A translational study showed that an overexpression of ERCC5 correlates with trabectedin activity and is associated with longer PFS in advanced STS treated with trabectidine [30]. Furthermore, in a cohort of STS, the frequency of SNP rs1047768 is the same as that of the general population, while the frequency of SNP rs2296147 is lower than that of the general population; the gene is overexpressed in 42% of the STSs analyzed and its expression correlates with that of the ERCC2 gene. Finally, the effect of SNP rs1047768 in protein structure was hypothesized for the first time in this study, suggesting a possible effect in ssDNA binding [14]. A meta-analysis associated variations on the ERCC5 gene with an increased risk of STS [31].

ERCC6 (CSB)

The encoded protein has ATP-stimulated ATPase activity, interacts with several transcription and excision repair proteins, and may promote complex formation at DNA repair sites. CSB interacts with RNAPII at the damaged site, and by direct interaction it recruits CSA [32], forming a complex responsible for the association and stabilization of UV-stimulated scaffold protein A (UVSSA), which stimulates TC-NER [33]. Mutations in this gene are associated with Cockayne syndrome type B and cerebro-oculo-facio-skeletal syndrome (COFS) [34]. On dbSNP are reported 68 clinically significant pathogenic variants of ERCC6 [35]. A retrospective translational study on STS showed that ERCC6 was underexpressed in L-sarcomas, compared with other STS subtypes [24].

ERCC8 (CSA)

CSA, encoded by ERCC8 (chr 10), is part of an E3-ubiquitin-ligase complex. CSA, in TC-NER, is required for recovery of DNA synthesis after repair is responsible for ubiquitination and proteasomal degradation of CSB, is required for recovery of DNA synthesis after repair [36] and interacts with CSB and with p44, a subunit of TFIIH. Mutations in this gene have been identified in patients with the hereditary disease of Cockayne syndrome (CS). [34], however genetic polymorphisms are shown to increase breast [37], gastric [38] and oral [39] cancer risk. On dbSNP are reported 32 clinically significant pathogenic variants of ERCC8 [35]. There are no data at present on the correlation of ERCC8 and STS.

XPA

The XPA protein is a zinc finger protein that plays a central role in NER by interacting with DNA and other proteins, forming the structure required to assemble the NER etching complex [40]. A retrospective translational study on STS showed that high levels of XPA expression correlated with better efficacy of trabectedin. [24].

XPC

It is a key component of the XPC complex, which plays an important role in the early stages of GG-NER. It has higher affinity for single-stranded DNA, and is important for damage detection [41]. At present, there are no experimental data on the role of XPC in STS.

TFIIH

TFIIH is a 10-subunit protein complex involved in both transcription and DNA repair, highly conserved in the entire eukaryotic domain. It can be divided in a 7-subunit CORE complex, consisting of XPB, XPD, p62, p44, p34, p52 and p8, and a CAK module (Cyclin Activated Kinase), comprised of CDK7, cyclin H and MAT1 [42]. XPB and XPD are both ATP-dependent DNA helicase and they catalyze the ATP-dependent opening of the DNA at the transcription starting site or at the damaged site. XPB, encoded by ERCC3 in chromosome 2, unwinds the DNA helix in the 3′-5′ direction and can also function as a 5′-3′ DNA traslocase [43], while XPD, encoded by ERCC2 in chromosome 19, acts in 5′-3′ direction and it’s responsible for recruiting the CAK complex [44]. The remaining 5 subunits (p62, p44, p34, p52 and p8) are encoded respectively by GTF2H1 in chromosome 11, GTF2H2 in chromosome 5, GTF2H3 in chromosome 12, GTF2H4 and GTF2H5 in chromosome 6; they carry out structural and ATPase regulation roles [45]. The three-subunit detachable CAK module has a fundamental role as a regulator of both transcription and damage repair pathways; in particular it is necessary for transcriptional activation, but its presence inhibits damage-repair functions [46]. Of the seven genes encoding the CORE components of TFIIH, mutations in ERCC3 and ERCC2 affect both RNA transcription and DNA repair pathway, causing severe disorders such as Xeroderma Pigmentosum, Cockayne Syndrome and Trichothiodystrophy [47]. NCBI dbSNP reports 14 clinically significant pathogenic or likely-pathogenic variants of ERCC3 [35]; in addition, various ERCC3 polymorphisms have been linked to increased risk of lung cancer [48] and osteosarcoma [49]. Regarding ERCC2, dbSNP reports 41 pathogenic or likely-pathogenic variants and polymorphisms in this gene have been associated with a higher risk of lung [50] and colorectal cancer [51]. Furthermore, a significant link has been reported between specific ERCC2 and ERCC3 SNPs and their predisposition to specific types of sarcomas [52]. The other 5 genes of the CORE complex are less affected by clinically significant polymorphisms, although it has been reported that mutations in p52, p8, and p44 are associated with developmental disorders [45]. Regarding the CAK module, high expression of cyclin H has been associated with trabectedin sensitivity in STS [24].

Ubiquitylation in NER Pathway

Ubiquitin is a 76-amino acid protein used for labeling targeted proteins, regulating their stability and function. Ubiquitylation is a sequential process that involves the action of E1 ubiquitin activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligating enzyme [53]. There are 2 E1, 40 E2 and about 600 E3 enzymes in the human genome [54], which emphasizes the specificity of the ligation process [55]. The initial ubiquitin is attached to the target protein in a lysine (K) residue in the C-terminal portion of the target. Subsequent ubiquitin molecules are sequentially attached to lysine residues of the previous molecule. Poly-ubiquitin chains can have different functions depending on the lysine residue to which the molecules link. The K48-linked chains signal proteasomal degradation of the target protein, whereas K63-linked chains regulate target protein function [56]. Ubiquitylation is reversible by the action of deubiquitinating enzymes (DUBs) [57]. Complex processes such as NER require similarly complex regulation through easily inducible and reversible post-translational modifications. Ubiquitylation has been shown to play a key role in this pathway [58].

GG-NER Regulation by Ubiquitylation

In the presence of UV damage to DNA, the COP9 signalosome is released from the CRL4^DDB2 complex, an E3 ubiquitin ligase comprising CUL4, ROC1, and DDB2. In normal conditions the COP9 signalosome inhibits the CRL4^DDB2 complex activity, in the absence of this inhibition CUL4 can be neddylated by NEDD8, leading to the activation of the E3 complex [59]. At this stage, recognition of DNA damage by XPC and DDB2 occurs [60]. The CRL4^DDB2 complex carries out the action of E3 ubiquitin-ligase on histones H2A, H2B, H3, and H4, weakening the histones-DNA interactions in the damaged area [61]. The complex auto-ubiquitinates DDB2, decreasing its affinity to damaged DNA [62]. This process competes with the presence of XPC at the damaged site, which stabilizes DDB2 [63], and with PARylation of DDB2 by PARP1, which inhibits its ubiquitination [64]. Deubiquitinase BAP1 also appears to be involved in this regulation process [65]. DDB2 is extracted from the complex by VCP/p97 and targeted to the proteasome. The removal of DDB2 from DNA increases the binding affinity of XPC to TFIIH, which is recruited at the damaged site. TFIIH promotes, through its p62 subunit, DDB2 extraction from the complex. XPC-TFIIH-XPA complex formation allows the initiation of the DNA damage verification process [66]. Simultaneously with DDB2 ubiquitylation, the CRL4^DDB2 complex also ubiquitinates XPC, not resulting in degradative signaling, but increasing its affinity to DNA [67]. XPC then undergoes SUMOylation, induced by UV damage to DNA, which results in the recruitment of RNF111/Arkadia (SUMO-targeted ubiquitin ligase), responsible for XPC ubiquitylation that leads to its removal from damaged DNA [68]. Extraction of XPC by VCP/p97 allows the other factors of GG-NER to be recruited.

TC-NER Regulation by Ubiquitylation

In the presence of DNA damage, RNA-Pol II is interrupted, recruiting CSA and CSB, which in turn recruit UVSSA. CSA is part of CRL4^CSA E3 ubiquitin-ligase complex, comprising CUL4, ROC1 and CSA. As in the GG-NER regulation, this complex is regulated by the COP9 signalosome, which detaches in the presence of DNA damage and allows the neddylation of CUL4, thereby activating the E3 complex [59]. CSB undergoes modification by multiple factors, as it is ubiquitylated by CRL4^CSA complex and BRCA1-BARD [69] and deubiquitylated by the deubiquitylating enzyme USP7 [70], which is recruited by UVSSA [71]. This fine-tuned regulation controls the stability of CSB before its extraction by VCP/p97, allowing the recruitment of the other damage repair factors. CRL4^CSA complex also mono-ubiquitylates UVSSA, allowing recruitment of TFIIH, which is then linked to RBP1 [60]. If the damage is not repaired, as in the case of mutations in CSA or CSB, the RBP1 subunit is ubiquitylated by NEDD4 [72] and the Elongin A ubiquitin ligase complex [73], inducing its extraction by VCP/p97 and subsequent proteasome degradation.

Role of Chromatin in NER Pathway

Activation of the NER pathway requires DNA not wrapped around histones, as various proteins need to access the double helix. UV damage to DNA provides the signal that leads to post-translational modifications of histones or ATP-dependent chromatin remodeling. These mechanisms allow the relaxation of chromatin around histones and increase the efficiency of the NER pathway [74,75].

Histone Modifications

Acetylation is mediated by histone acetyltransferases (HATs) and the reverse process, deacetylation, is catalyzed by deacetylases (HDACs). Histone acetylation promotes chromatin relaxation and activation, promoting DNA transcription [76]. Histone acetylation stimulates NER after UV damage [65]. Recent studies report that DDB2 interacts with HBO1 (HAT) in a UV-dependent manner leading to acetylation of H3 and H4, which increases chromatin accessibility [77]. DDB2 appears to be responsible for deacetylation of H3 and H4 through HDACs, leading to the stimulation of XPC recruitment [78]. Methylation acts differently on chromatin state depending on the number of methyl groups and the residues on which they are added [79]. Recent work has shown that DDB2 recruits the methyltransferase ASH1L to damaged DNA regions, leading to tri-methylation of histone H3K4. This process induces XPC binding to nucleosomes [80]. UV irradiation also stimulates tri-methylation of H3K79 by DOT1L, which also correlates with XPC recruitment. Deletion of DOT1L is present in many cases of melanoma [81]. Finally, another histone modification is phosphorylation which leads to a more relaxed state of chromatin and serves as a checkpoint in several processes, including DNA repair [82]. Phosphorylation of specific histones does not appear to affect the NER pathway; rather, it is the NER pathway that induces phosphorylation of histone H2AX via single-strand DNA production [83].

ATP-dependent Chromatin Remodeling

Chromatin remodelers are enzymes with an ATPase domain, which use ATP energy to modify the structure of nucleosomes. They are divided into 4 families: SWI/SNF, CHD, INO80, ISWI [84]. During NER, the SWI/SNF complex catalyzes the relaxation of chromatin, making it more accessible. CSB, which plays a key role in the NER pathway, belongs to this group [85]. An additional role of SWI/SNF is its association with XPC, which promotes the recruitment of subsequent repair factors [86]. The remodeler INO80 interacts at the level of damaged sites with DDB1, suggesting a role in XPC recruitment [87]. CHD is recruited following UV damage and mediates XPC binding to TFIIH [88].

Histone Chaperones

Proteins are involved in the transport and mobilization of histones at the chromatin level [89]. CAF-1 and HIRA are associated with NER, as they are recruited in the late stages of the pathway and are involved in the deposition of neo-synthesis histones after damage repair [90,91].

Conclusions

DDR pathways, including NER, play a key role in the formation of various tumor types and their sensitivity or resistance to treatment. The NER pathway is composed of a variety of processes that are finely regulated by each other and integrated with many other cellular pathways and alterations in this pathway play an important role in many tumor types, including STS. Recent studies contribute to the notion that NER pathway deficiencies constitute potential cancer therapeutic targets. It has been found that putative damaging germline and somatic alterations in NER genes were present in STS [14]. Moreover, recent findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs [9]. There is still little evidence on the association between cancer risk and variations in NER genes; in fact, there are no FDA-approved targeted therapies that target germline or somatic mutations in NER pathway genes. However, mutations in NER genes can have multiple roles as biomarkers: they can act as predictive biomarkers, indicating an increased risk of developing cancer, being useful for early cancer detection by subjecting the population to higher levels of screening; at the same time, they can also be used as prognostic biomarkers, giving precise indications to physicians about the degree of sensitivity to drugs targeting DNA repair deficiencies, if variations in NER genes are present. Recent studies focus instead on the therapeutic role of NER inhibitors, such as spironolactone [92] well as triptolide, which inhibits NER by affecting XPB and transcription. NER inhibition has been shown to reverse acquired resistance to alkylating agents in multiple myeloma cells [12]. Hence, it may be another adjunct target to be considered in combination therapies. Their further investigation in these tumor types is necessary for the identification of new biomarkers or therapeutic targets (Figures 1 and 2).

Figure 2: Nucleotide excision repair pathway (NER). NER pathways can be classified as either global genome repair (GGR) and transcription-coupled repair (TCR). The NER pathway consists of a series of reactions: recognition of DNA damage, unwinding double-stranded DNA in the neighborhood of the damage, excision of the damaged nucleotides, and filling the gap by DNA synthesis and ligation.

Author Contributions

Conceptualization, S.P.; writing—original draft preparation, F.S., A.P., A.B., E.C, S.F., O.C., E.G. and S.P; writing—review and editing, I.P, A.B. and G.S.; supervision, A.L. and S.P.; funding acquisition, I.P., D.A.C, S.P.; figures preparation: S.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Regione Toscana Bando Salute 2018, (Research project CUP n. D78D20000870002), grant number D78D20000870002.

Acknowledgments

Our special memory goes to Alessio Cerasola, an unforgettable guy who fights the disease with his ever-present smile.

Conflicts of Interest

The authors declare no conflict of interest

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DOI: 10.31038/CST.2023822

Abstract

Gastric cancer (GC) is one of the three most deadly among cancers. Although several new drugs have been introduced for metastatic disease, the median overall survival (OS) remains 11-14 months. Perioperative chemotherapy (CT) is the current standard of care for resectable cT2-4 and/or N+ GC, and FLOT (5-fluorouracil, oxaliplatin, docetaxel) is the treatment of choice. To date, no predictive factor of response has been identified. Considering their synergism in DNA repair, ataxia telangiectasia mutated (ATM) and sirtuin-1 (SIRT1) merit investigation for prognostic stratification.

We evaluated by immunohistochemistry the expression levels of ATM and SIRT1 in the surgical specimens from 42 patients with a resectable GC or gastroesophageal junction adenocarcinoma treated with neoadjuvant CT and surgery. In the entire population, median DFS was 22.2 months (95%CI 14.9 – NR) and median OS was not reached (95%CI 26.9 – NR). DFS was significantly longer in patients achieving tumor regression grade (TRG) 2-3 compared with those achieving TRG 4-5 (median DFS not reached vs. 14.9 months; HR=0,36 CI=0,14-0,97; p=0.034), and a trend toward a better OS was also observed across the two subgroups (p=0.068). The proportion of patients who obtained a major/medium pathological regression was higher in the ATM-absent group than in the ATM-expressed group (69% vs. 50%; X-squared=6.05; p=0.1). In the overall population, OS and DFS did not have a significantly different distribution according to ATM and SIRT1 expressions. In contrast, in the TRG 4-5 subgroup, the ATM expression seems to be associated with inferior DFS (7.7 months vs. 32.1 months; p=0.055), particularly when combined with absence of SIRT1. In conclusion, TRG has been confirmed a surrogate of survival, ATM expression correlates with TRG and TRG-high/ATM-expressed/SIRT1-absent profile should be studied as a prognostic marker in prospective trials.

Keywords

Gastric cancer, Prognostic biomarker, Predictive biomarker, Epigenetic, DNA damage repair, ATM, SIRT1, TRG, DNA double-strand break

Key Points

1. No prognostic markers are available in gastric cancer patients after neoadjuvant chemotherapy.
2. TRG is a surrogate of survival outcomes.
3. TRG-high/ATM-expressed/SIRT1-absent profile should be prospectively investigated as a marker for prognostic stratification.

Introduction

Gastric cancer (GC) is one of the three most deadly among cancers, and the fifth most commonly diagnosed tumor worldwide, with about 950,000 new cases per year [1]. Although several new drugs have been introduced for metastatic disease, the median overall survival (OS) remains 11-14 months [2,3]. In recent years, perioperative chemotherapy (CT) has been considered the standard of care for the vast majority of patients with resectable GC, and FLOT (5-fluorouracil, oxaliplatin, docetaxel) is the treatment of choice in patients fit for intensive CT. Doublets containing fluoropyrimidine and platinum are considered feasible for frail, comorbid or elderly patients [4-6]. To date, no reliable predictive factor of benefit from perioperative treatments has been identified and a tailored strategy is yet to be applied [7]. An appropriate patient selection for perioperative therapy is challenging, since only a few features have been associated with tumor regression and survival outcome after FLOT treatment. In the FLOT4-AIO trial, diffuse histotype has been associated with a reduced rate of pathological complete response (pCR) compared to the intestinal one (3% vs. 23%, respectively). Among the tumor-centered endpoints, pCR is currently considered of great interest as it could be a surrogate for survival outcomes [8]. Unfortunately, in resectable disease, only limited evidence is available regarding a molecular biomarker-based patient selection [7]. Although several molecular subgroups have been identified as potentially associated with a prognostic or predictive effect, stratification in prospective trials is still needed. Among the putative biomarkers of interest, microsatellite instability (MSI) seems to be the best candidate to drive treatment choice in the near future [7,9,10]. Consistent with previous evidence, a recent international meta-analysis conducted by Pietrantonio et al. confirmed MSI-high (MSI-H) status as a favorable prognostic factor in patients with resected GC. The 5-year disease-free survival (DFS) and overall survival (OS) rates were higher in patients with MSI-H GC than in those with microsatellite stable (MSS) tumor (5-year DFS: 71.8% vs. 52.3%, respectively; 5-year OS: 77.5% vs. 59.3%) [9]. In addition, the benefit from neoadjuvant CT in resectable MSI-H GC seems to be limited, and that ongoing studies are evaluating immune checkpoint inhibitors (ICIs) as neoadjuvant therapy or potentially curative strategy in patients achieving a complete clinical-pathological-molecular response [10]. Although evidence is limited, ataxia telangiectasia mutated (ATM) protein and sirtuin-1 (SIRT1) have demonstrated a deep synergism of action and could be considered for prognostic stratification, since they are involved in the repair of DNA double-strand breaks (DSB) and epigenetic regulation [11-13].

The ATM gene is located on chromosome 11 and encodes a serine/threonine protein kinase that contributes to maintaining genomic integrity transducing a DSB repair signal to effectors. ATM protein levels are decreased in GC compared to normal samples and low levels of phosphorylated ATM are associated with poor differentiation, lymph node metastasis and poor prognosis [14]. GC cells with defective ATM (expression or activity) determining homologous recombination deficiency are more sensitive to therapies that cause the accumulation of DNA DSBs [15]. In particular, in GC cell lines, ATM overexpression is associated with cisplatin-resistance and its inhibition, using the ATM inhibitor CP466722 or siRNA, induces the reversion of epithelial-to-mesenchymal transition [11]. Therefore, mediating platinum-resistance, we supposed that ATM expression could influence tumor regression in GC patients treated with platinum-based neoadjuvant CT.SIRT1 is a NAD+-dependent class-III histone deacetylase (HDAC) involved in several cell functions including DNA repair. Contributing to the identification of DNA damage sites and access of DNA repair proteins, SIRT1 has a crucial role in the epigenetic regulation of cell homeostasis by deacetylating both histone and non-histone proteins. SIRT1 acts both as tumor suppressor and tumor promoter, depending on location (nucleus vs. cytoplasm) and tissue type. DNA damage is a trigger for SIRT1 dissociation and re localization to DSB. In the SIRT1-DNA repair interplay, ATM preserves the efficient recruitment of SIRT1 to DSBs by signaling DNA damage. Simultaneously, SIRT1 stabilizes ATM at DSB sites and stimulates its autophosphorylation and activity [12,13].

The aim of this study was to investigate the prognostic role of ATM and SIRT1 expression in a cohort of patients who underwent neoadjuvant CT for resectable GC.

Materials and methods

Study Design and Population

This was a single-institution, observational, retrospective and prospective study, which enrolled patients with a resectable GC or gastroesophageal junction adenocarcinoma treated with neoadjuvant CT and surgery from September 2017 to April 2022.

Being an observational study, treatments were performed according to clinical practice and national and international guidelines, regardless of the inclusion in the study.

All patients had a histological diagnosis of GC or gastroesophageal junction adenocarcinoma and availability of tissue samples of primary tumor for translational analysis. Clinical stage was assessed in a multidisciplinary board according to the 7th edition of the International Union Against Cancer Tumour–Node–Metastasis classification [16]. A CT scan, FDG-PET scan, US-endoscopy and, wherever indicated, diagnostic laparoscopy, were routinely performed for staging. Patients with metastatic disease, squamous cell carcinoma, pure neuroendocrine carcinoma or esophageal cancer were excluded.

The primary objective was to describe the expression of ATM and SIRT1 in a cohort of patients with resectable GC who had received neoadjuvant CT. Secondary objectives included the description of clinical characteristics associated with specific patterns of expression and the identification of clinical and/or pathological characteristics that may be related to prognosis.

This study was approved by the Institutional review board of Azienda Ospedaliero-Universitaria Careggi (Comitato Etico Regionale for clinical experimentation of Toscana Region – Italy – Area Vasta Centro – 22070_BIO). Informed consent from each patient enrolled in the study was obtained.

Histopathological Evaluation and Immunohistochemical Staining

Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded (FFPE) tumor sections of GC, 3µm thick. Tissue sections were processed by fully automated detection and staining techniques through Discovery Ultra immunostainer (Ventana Medical Systems, AZ, USA). Slide sections were incubated with the following primary antibodies: anti-SIRT1 (#ab104833; mouse monoclonal, clone 1F3, 1:500, Abcam, Cambridge, UK) and anti-ATM (#ab32420; rabbit monoclonal, clone Y170, 1:100, Abcam, Cambridge, UK). Anti-SIRT1 signals were developed with UltraMap anti-mouse HRP, and anti-ATM with UltraMap anti-Rabbit HRP (Ventana Medical Systems, AZ, USA). The bound antibodies were visualized using Discovery ChromoMap DAB Kit (Ventana Medical Systems, AZ, USA). Finally, sections counterstaining was performed with Haematoxylin II ready-to-use; Ventana, AZ, USA). Healthy human colon for SIRT1 and testis for ATM were used as positive controls. Negative control was performed by replacing the primary antibodies with Mouse IgG1 and Rabbit IgG Isotype Control (Invitrogen). The negative and positive control sections were treated in parallel with the samples in the same run Immunohistochemical expression of SIRT1 was evaluated according to An et al. [17].

The expression score was assessed by combining staining intensity score and the positive percentages. The expression was scored as follows: <10%, 10-24%, 25-49%, 50-74%, and ≥75%. Immunohistochemical expression of ATM was evaluated according to Miller et al. [18]. The staining was evaluated based on nuclear DAB signal, and the intensity score was assessed as: 0 to 3, scaled in 0.25-point increments (0=totally negative; +/3=weak positive; ++/3=moderate positive; +++/3=strongly positive).

Statistical Analysis

Demographic and clinical data, molecular alterations, disease and treatment characteristics were analyzed using descriptive statistics. Statistical comparisons for categorical variables were performed using X2 test. Time-to-event endpoints were described by Kaplan-Mayer curves. Survival distributions for specific subgroups of patients were tested with the log-rank test. A p-value of 0.05 or lower was considered statistically significant. All the analyses were corrected for multiple testing when appropriate and challenged with comprehensive multivariate modeling.

Results

Between September 2017 and April 2022, a total of 42 patients were prospectively and retrospectively enrolled. Twenty-nine were men (69%) and 13 were women (31%) with a median age of 68 years (range 49-79). The primary tumor location was corpus-antrum in 79% of cases and gastroesophageal junction in 21%. A vast majority of the patients (90%) had an optimal performance status (ECOG PS0) at the time of the initial diagnosis. Among the most common presenting symptoms, weight loss more than or equal to 5 kg was reported in 19%. As neoadjuvant treatment, 79% (n=33) of patients had received a taxane-based triplet, while 21% (n=9) received a taxane-free doublet. Postoperative CT was administered in only 50% of cases, due to suboptimal recovery from surgery or postoperative complications, and 85% of them required a dose reduction due to toxicities. In the surgical specimens, lymph node involvement was reported in 50% (n=21) of patients and pT3-4 in 64% (n =27). Clinico-pathological characteristics are summarized in Table 1.

Table 1: Clinical and pathological characteristics of patients

At the time of the analysis, 16 patients were deceased and 26 patients were still living. Disease recurrence occurred in 40% of patients (n=17). Median DFS was 22.2 months (95%CI 14.9 – NR) and median OS was not reached (95%CI 26.9 – NR). Among the other clinico-pathological factors, univariate analysis showed that weight loss at diagnosis (p=0.03), pathological nodal involvement (p=0.002) and number of neoadjuvant cycles (p=0.03) were significantly associated with DFS. In addition, ypT (p=0.05), ypN (p=0.007), number of neoadjuvant cycles (p=0.0018) and number of adjuvant cycles (p=0.008) were significantly associated with OS. The multivariate analysis confirmed the association between the number of adjuvant cycles and DFS (p=0.018) and OS (0.023) and between the number of neoadjuvant cycles and OS (p=0.042).

A histopathological review was performed by dedicated pathologists focusing on the assessment of tumor regression grade (TRG) according to Mandard. As reported in Table 1, TRG 2 was reported in 24% of cases (n=10) and TGR 3 was reported in 33,3% (n=14), while both TGR 4 and TGR 5 were described in 19% of cases (n=8). Then, we assessed whether TRG was associated with survival outcomes. Consistent with the literature, DFS was significantly longer in patients achieving TRG 2-3 compared with those achieving TRG 4-5 (median DFS not reached vs. 14.9 months; HR=0,36 CI=0,14-0,97; p=0.034) and a trend toward a better OS was also observed across the two subgroups (median OS not reached vs. 26.9 months; HR=0,39; CI=0,14-1,11; p=0.068) (Figures 1 and 2).

Figure 1: DFS in GC patients achieving TRG 2-3 vs TRG 4-5

Figure 2: OS in GC patients achieving TRG 2-3 vs TRG 4-5

We evaluated the expression of ATM and SIRT1 in the surgical specimens by IHC. Although optimal ATM staining cutoffs were controversial, according to Kim et al. the criteria for negative cases were set as less than 10% of cells stained as weak positive (+/3) or higher intensity, that is, more than 90% of cells showing totally negative (0) or equivocal staining (±) [15]. For example, if more than 90% of tumor cells showed equivocal (±) or negative (0) staining and less than 10% showed any positive (+, ++ or +++/3) staining, a case was defined as negative (Figure 3).

Figure 3: Representative H&E images and IHC of ATM and SIRT-1 expression in GC specimens. Representative images of GC patient with 0 ATM score, 3 SIRT1 score, and TRG of 2 (A); representative images of GC with 3+ ATM score, 0 SIRT1 score, and TRG of 5 (B); representative images of GC with ATM and SIRT1 score 0, and TRG of 5 (C); representative images of GC with 0 ATM score, 1 SIRT1 score, and TRG of 4 (D); (Magnification ×20, inset ×40; scale bar 100 μm, 50 μm, respectively).

Accordingly, as reported in Table 2, ATM score was 0 in 16/42 cases (38%), 1+ in 16/42 (38%), 2+ in 7/42 (17%) and 3+ in 3/42 (7%).

Table 2: ATM and SIRT1 expression

SIRT1 expression was <10% in 74% of cases, 10-24% in 7%, 25-49% in 10%, 50-74% in 7% and ≥75% in 2% [12]. We then we evaluated the correlations between the expression of ATM and SIRT1 and TRG. Of note, the proportion of patients who obtained a major/medium pathological regression (TRG 2-3) was higher in the ATM-absent subgroup than in the ATM-expressed subgroup (69% vs. 50%; X-squared=6.05; p=0.1) (Figure 4).

Figure 4: Mosaic plot including TRG and ATM as variables

In the overall population, OS and DFS did not have a significantly different distribution according to ATM and SIRT1 expressions (OS p=0.4 and p=0.2, respectively; DFS p=0.56 and p=0.81, respectively).

In contrast, in the subgroup of patients with TRG 4-5, usually characterized by poor prognosis, the absence of ATM expression seemed to be a positive prognostic factor. The median DFS in patients whose tumor had TRG 4-5 and absent ATM expression was 32.1 months compared to 7.7 months in those with TRG 4-5 and positive ATM expression (p=0.055). Although not statistically significant, the median OS was numerically higher in the subgroup with ATM-absent than in the subgroup with ATM expression (55.0 months vs. 26.9 months, respectively; p=0.6) (Figures 5 and 6).

Figure 5: DFS distribution in patients whose tumor had TRG 4-5 according to ATM expression

Figure 6: OS distribution in patients whose tumor had TRG 4-5 according to ATM expression

Furthermore, among patients with TRG-high and ATM-expressed, those with SIRT1 <10% had a median DFS of 12.7 months, which was significantly inferior if compared with the entire population (median DFS not reached; HR=0.31; CI=0.11-0.91; p=0.024) (Figure 7).

Figure 7: DFS distribution between TRG-high/ATM-expressed/SIRT1-absent profile vs other profiles

This difference was partially sustained by the positive prognosis of TRG-low patients. Therefore, excluding patients with TRG-low, the TRG-high/ATM-expressed/SIRT1-absent profile was associated with a trend toward a lower DFS compared with other TRG-high patients (median DFS 12.7 months vs. 32.1 months; p=0.32) (Figure 8).

Figure 8: DFS distribution between TRG-high/ATM-expressed/SIRT1-absent profile vs TRG-high subgroup

Discussion

Since the perioperative treatments have become the standard of care for a vast majority of patients with cT2 or higher and/or nodal-positive resectable GC, the identification of new prognostic and predictive biomarkers is an urgent need. ATM expression has been extensively studied with conflicting results [19-21]. Kelmpner et al. did not show any association between ATM expression and clinico-pathological factors and any impact on prognosis from ATM profiles in a cohort of patients who were treated with first-line XELOX for advanced GC [20]. In contrast, in a study of Kim et al., a low ATM expression was associated with older age, advanced stage, MSI, and lower DFS and OS in patients who underwent radical surgery for resectable GC. In this study, the worst prognosis was exhibited by the subgroup which had low ATM expression and MSS [21]. Although the setting was similar to our study, the patient population of Kim and colleagues received upfront surgery followed by adjuvant CT in 50% of cases, while all our patients received neoadjuvant CT followed by surgery, and this may have contributed to a different ATM expression and prognosis. In our study, patients with ATM-expressed cancer after neoadjuvant CT were more frequently associated with TRG 4-5 and, consequently, had a worse prognosis. Although the real reason remains largely unknown, we can suppose that high expression of ATM, playing a crucial role in the repair of DSBs, may offer a highly efficient mechanism of repair from damages induced by CT, radiation, oxidative stress, and stochastic events [11]. As previously described, repair of DSBs involves an extensive network of signals, including a synergism between ATM and SIRT1 with epigenetic implications [11-13]. To date, the role of epigenetic alterations and changes during CT is debated. An extensive knowledge of epigenetic mechanisms underlying prognosis and treatment response could produce new promising epigenetic strategies for GC treatment [22]. SIRT1 contributes to several processes involving GC development, invasion, and metastatic spread. In preclinical studies, knockdown of SIRT1 promoted GC cell migration and invasion in vitro and metastasis in vivo. Among genes downregulated by SIRT1, ARHGAP5 has been identified as an independent prognostic marker of GC [23-25]. As previously described, SIRT1 has an ambiguous role acting both as tumor suppressor and tumor promoter [12,13]. An et al. explored the role of SIRT1 expression in chemoresistance of GC both in vitro and in vivo. They showed that SIRT1 had inhibitory activity on chemoresistance and eliminated cancer stem cell properties [17]. Several retrospective studies suggested a negative prognostic impact of SIRT1-high profile than SIRT1-low, but the prognostic role of SIRT1 expression remains unclear. In a study by Noguchi et al, patients with SIRT1-high GC had a shorter cancer-specific survival than patients with SIRT1-low GC [26,27]. Similarly, Zhang et al. showed that low SIRT1 expression was associated with better outcomes both in patients with advanced GC and in those with early-stage GC [28]. In contrast, Kang et al. reported a positive prognostic effect from SIRT1 expression in a cohort of 452 patients who received surgery for GC. In this study, SIRT1-high profile was associated with more favorable clinicopathological features, including intestinal histotype, lower grade, and lower pT and pN stage [29]. In our study, among patients with poor prognosis (TRG-high and ATM-expressed), a numerically lower DFS was observed in the SIRT1 <10% group than in the SIRT1 ≥10% group. Although statistical significance was not reached, we can speculate that a complete loss of SIRT1 expression could be associated with inhibition in reversing chemoresistance and amplification of the negative prognostic effect of ATM-high profile which efficiently repairs chemotherapy-induced DNA damage. This hypothesis is consistent with a study by An et al. [17], in which silencing of SIRT1 facilitated resistance to 5-fluorouracil and cisplatin.

Regarding limitations, our study included only patients of European origin and the comparison with studies carried out in Asia might be precluded by the geographic heterogeneity in pathological features of GC. In addition, the limited sample size could have induced us to underestimate small differences or subgroup effects. Availability of pretreatment biopsies would have contributed to a better interpretation of the results.

In conclusion, the role of ATM and SIRT1 expression in resected GC patients has not been thoroughly explored and could be of interest for the generation of hypotheses that warrant a future prospective validation in larger clinical trials. This study confirms TRG as a surrogate of survival and suggests an association between ATM expression and TRG. The TRG-high/ATM-expressed/SIRT1-absent profile tends to be associated with a poor prognosis and merits study as a stratification marker after neoadjuvant CT.

Funding

No financial funding was received.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article.

Competing Interests

The authors declare that they have no competing interests.

Abbreviations

ATM: Ataxia Telangiectasia Mutated; CT: Chemotherapy; DFS: Disease-Free Survival; FFPE: Formalin Fixed Paraffin-Embedded; FLOT: 5-Fluorouracil, Oxaliplatin, Docetaxel; GC: Gastric Cancer; HDAC: Histone Deacetylase; IHC: Immunohistochemistry; MSI: Microsatellite Instability; MSI-H: Microsatellite Instability-High; MSS: Microsatellite Stable; OS: Overall Survival; pCR: Pathological Complete Response; SIRT1: Sirtuin-1; TRG: Tumor Regression Grade

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