OC is a misleading term for a series of genomically and etiologically heterogenic diseases that often do not arise from ovarian tissue and simply share an anatomic location, resulting in different OC histotypes (i.e., mucinous, endometrioid, clear cell, and serous)[3, 14, 15]. Hence, given the complexity of OC, the current approaches of treating OC as a single disease has to move toward patient-tailored therapy based on molecular and histotype-driven treatments (biotypes). Recently, The Cancer Genome Atlas (TCGA) Research Network (http://cancergenome.nih.gov/) described an integrated analysis of the genomic and epigenomic landscapes of almost 500 high-grade serous OC patients with advanced stage disease, with the aim to identify molecular abnormalities that influence pathophysiology and outcomes and could constitute therapeutic targets[16]. This integrated analysis has definitely demonstrated the low mutation rate of high-grade OC, with the exception of the tumor protein 53 (TP53) and breast cancer genes 1 and 2 (BRCA1/2), which are affected in 96% and 22% of OC cases, respectively. In contrast, extensive focal and broad DNA losses and gains were observed through genomic analyses of OC with DNA deletions and amplifications affecting many genes. Interestingly, germline mutations in the BRCA1/2 genes are significantly associated with improved OS and overall response rate (ORR) to platinum-based chemotherapy[16–19]. Indeed, deficiencies in the homologous recombination pathway, which is involved in DNA repair, can disrupt the repair of DNA crosslinks introduced by platinum-based chemotherapy and other DNA-damaging agents, such as trabectedin, resulting in higher survival rates due to an improved response in BRCA-deficient patients. Furthermore, these homologous recombination deficiencies sensitize tumors for targeted therapies such as poly(ADP-ribose) polymerase (PARP) inhibitors[20] or trabectedin[21], as we will discuss later.

Nucleotide excision repair (NER) is the primary mechanism known to remove platinum-DNA adducts (including cisplatin-induced intra-strand crosslinks) from DNA[42], thus increasing resistance to the drug. NER is composed of two subpathways for DNA damage detection: 1) global genome NER, which maintains the integrity of the entire genome, and 2) transcription-coupled NER, which accounts for removal of damage in the transcribed strands of active genes. Impaired NER activity is associated with several rare autosomal-recessively photosensitive diseases, including xeroderma pigmentosum (XP), Cokayne syndrome, and trichothiodystrophy [43]. NER factors are also mutated in some human cancers, including ovarian tumors[44], and can therefore contribute to clinical resistance to platinum derivates[42].

Global genome NER and transcription-coupled NER are comparable in terms of mechanism, except for the initial damage-recognition step[43]. In global genome NER, helix-distorting base lesions are identified by xeroderma pigmentosum group C and RAD23 yeast, homolog of B proteins (XPC-hHR23B). In transcription-coupled NER, elongating RNA polymerase II encounters the lesion and recruits the Cokayne syndrome factors CSA and CSB. Subsequent stages of the two subpathways are the same. Transcription factor II human (TFIIH) is composed of xeroderma pigmentosum complementation group B (XPB) and D (XPD) subunits, which are helicase components that catalyze ATP-dependent unwinding of the DNA strands. XPA and replication protein A stabilize the open complex and position the other factors. Dual incision is then performed by the excision repair cross-complementation group 1 (ERCC1)-XPF and XPG endonucleases, which cleave the damaged strand 5′ and 3′ of the lesion, respectively. Finally, NER is completed by gap-filling DNA synthesis, followed by strand ligation (Figure 1 )[45].

As for all platinum-based drugs, DNA distortion produced by trabectedin is a substrate for the NER pathway; however, the mechanism by which trabectedin interacts with DNA and exerts its antitumor activity is different from those of cisplatin. Cisplatin binds to the N7 position of guanine in the major groove of DNA[42], whereas trabectedin, as previously mentioned, forms monoadducts at the N2 position of guanine in the minor groove of DNA, forcing the minor groove toward the major groove and allowing possible interactions between the C-ring portion of the drug and NER factors[23,25]. Initial studies revealed a unique pattern of sensitivity to the drug in cells with defects in the NER machinery[46–49]. It has in fact been reported that NER-deficient cells are significantly less sensitive to trabectedin (from 2- to 8-fold) than NER-proficient cells. This is unusual, intriguing, and somewhat paradoxical, as a deficiency in NER is generally associated with increased sensitivity to DNA-interacting agents[50]. The formation of lethal single-strand DNA breaks was reported to be related to a functional transcription-coupled NER pathway [49,51]. Soares et al.[52] performed an in vitro study and suggested that the action of trabectedin in eukaryotic cells might be the result of NER inactivation through the formation of an inactive XPG/DNA trabectedin ternary complex, which conveyed the idea that NER components may represent the primary targets of the drug. In this regard, using a reverse-phase lysate microarray platform, Stevens et al.[53] demonstrated, in a series of tumor cell lines, that XPF and XPG showed the highest negative correlations with drug activity across all three tissue-of-origin groups (colon cancer, OC, and XP cell lines). Furthermore, the increased XPF and XPG protein levels were directly correlated with trabectedin sensitivity and cisplatin resistance, as previously reported[48,54]. Despite this finding, the authors concluded that it is difficult to identify a single NER biomarker to predict drug sensitivity and that the heterogeneity of tumors and the influence of NER-independent pathways probably contribute to this complexity.

It has been shown that trabectedin-induced DNA damage requires double-strand base (DSB) repair, suggesting a pivotal role of this pathway in drug-induced cytotoxicity[52]. Hence, the homologous recombination repair of DSBs is also critical for modulating the cellular response to trabectedin in homologous recombination-deficient cells displaying hypersensitivity to the drug[21,52]. In the proposed model, the trabectedin adducts in the minor groove are recognized by the NER system, particularly by the XPG protein. The catalytic endonuclease activity of XPG was found to be dispensable, but its C-terminal region was essential for the formation of “cytotoxic complexes” that, during the S phase, gave rise to DSB lesions that had to be repaired by homologous recombination. Cells lacking the homologous recombination pathway are indeed extremely sensitive to the drug, with a decrease in the 50% inhibition concentration (IC₅₀) by approximately 100 folds[21]. In addition, complementation of the defects almost completely reverts the sensitivity to the normal control level, strongly suggesting that homologous recombination is a key determinant for repairing trabectedin-induced lesions[21].

Trabectedin treatment induces the formation of both phosphorylated histone H2AX (g-H2AX) and Rad51 foci in NER-proficient cells, suggesting that the DSBs in DNA are tentatively repaired by the induction of Rad51 foci. Both events are much less evident in a NER-deficient background, suggesting that, in the presence of trabectedin, the NER pathway is responsible for the incision/excision of the DNA lesion, leading to the formation of DSBs[21]. In addition, the induction of g-H2AX phosphorylation is much more evident in the S phase of the cell cycle, corroborating the hypothesis of a strict requirement for DNA synthesis in the formation of trabectedin-induced DSBs[52]. The fact that a clear-cut induction of g-H2AX phosphorylation and Rad51 focus formation is observed only in NER-proficient cells can be explained in light of the experiments performed by Tavecchio et al.[21] in S. pombe, where the formation and stabilization of a ternary complex among DNA-bound trabectedin and the Rad13 carboxy-terminal region was responsible for the toxicity of the drug. This suggests that the NER proficiency leads to the accumulation of unprocessed single-strand bases (SSBs) that can be converted to DSBs in the S phase. In contrast, the inability of NER-deficient cells to efficiently induce Rad51 foci appears to have a protective role when cells are treated with trabectedin[21].

The accumulating evidence of the existence of a subset of tumors with specific defects in the homologous recombination DNA repair pathway, the so-called BRCAness phenotype[55,56], clearly suggest that patients whose tumors harbor those specific defects should benefit more from a trabectedin-based therapy.

In an attempt to clarify these observations, Monk et al.[57] evaluated the role of 13 proteins, which are involved in cell proliferation (Ki67), cell cycle checkpoint signaling [P53, ataxia telangiectasia mutated (ATM), checkpoint kinase 1 (CHK1), and CHK21, and DNA repair [BRCA1, BRCA2, DNA-PK, excision-repair complementing defective in Chinese hamster 1 (ERCC1), Fanconi anemia complementation group D2 (FANCD2), Nibrin, Rad50, and XPA1, as potential predictive biomarkers in patients with advanced OC using the OVA-301 phase III trial series as the study population. The authors found that low protein expression of Nibrin was statistically associated with better clinical outcome (higher ORR in univariate analysis as well as longer PFS and OS in both univariate and multivariate analyses). Nibrin also showed a high predictive value for treatment outcome in patients with platinum-sensitive disease and in those who were treated with trabectedin plus PDL, but not in those treated with PDL alone.

Nibrin is a 754-amino acid polypeptide involved in the recognition and repair of DSBs[58]. This protein interacts with meiotic recombination 11 (MRE11) and RAD50 homolog (RAD50) to form the Mre11-Rad50-Nibrin (MRN) complex and is required for translocating this complex to the sites of DSBs[58]. The MRN complex plays a crucial role as a sensor of DSBs, in activating the signal transduction cascades that lead to cell cycle checkpoints and in regulating the DNA repair pathway selection.

From the beginning of its development, trabectedin has shown some peculiar properties that clearly distinguish it from other anticancer drugs. Trabectedin not only induces DNA damage, as already discussed, but also directly inhibits growth, induces cell death and differentiation of malignant cells, and affects the tumor microenvironment by reducing the production of key inflammatory mediators[26,59–61] (Figure 2 ).

It has been reported that trabectedin affects gene transcription through different mechanisms. First, trabectedin prevents the binding of transcription factors to DNA[62]. Second, the DNA adducts formed by trabectedin functionally mimic an inter-strand crosslinking lesion, which blocks transcription by stabilizing double-strand DNA[63]. Finally, trabectedin directly interacts with the elongating Pol II, regardless of the strands in which they are located[63].

In addition to its transcriptional action, trabectedin has selective anti-inflammatory and immunomodulatory properties on monocytes and tumor-associated macrophages (TAMs), and it inhibits the production of factors potentially relevant for tumor growth, tumor progression, and the inhibition of tumor-promoted angiogenesis. Indeed, trabectedin also targets inflammatory cells in the tumor microenvironment to reduce the angiogenic and pro-inflammatory effects mediated by cytokines, thus leading to a delayed response with prolonged stabilization (i.e., tumor dormancy). For example, trabectedin treatment down-regulates the expression of interleukine-6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-X-C motif) ligand 8 (CXCL8), angiopoietin-2, and vascular endothelial growth factor (VEGF), but not other biological mediators such as the tumor necrosis factor alpha (TNFα)[61,64]. The anti-inflammatory effects of trabectedin were confirmed in different tumor xenograft models and in human soft-tissue sarcoma samples from patients receiving trabectedin as neoadjuvant therapy[60].

Hence and overall, trabectedin is probably more than a cytotoxic drug, given that its antitumor activity arises from different combinations that act through more than one mechanism, and provides a consolidated therapeutic approach as a multitarget drug with far more multifaceted activity than initially formulated. Through these mechanisms, trabectedin is likely to impact relevant biological pathways involved in cancer, which may influence disease outcome.