The primary aim of this study was to establish a digital droplet PCR (ddPCR) methodology for accurately quantifying the concentration of extrachromosomal circular DNA (eccDNA) and to assess the effects of various chemotherapeutic agents on the copy number of WWP1-eccDNA. The results from both the linearity and reproducibility experiments have unequivocally demonstrated that ddPCR offers significantly greater sensitivity and accuracy than quantitative PCR (qPCR). Notably, ddPCR achieved stable detection at as low as 5 copies per microliter, in stark contrast to qPCR, which only achieved stable detection at concentrations starting from 100 copies per microliter, as illustrated in Figs. 2 and 3. These findings underscore the substantial benefits of ddPCR in oncological research, particularly in studies involving low-abundance genetic targets.
Furthermore, this research has pioneered the use of the ddPCR technique to detect genetic mutations in preserved samples from asymptomatic women at risk of developing ovarian cancer [13]. This innovative application highlights the potential of ddPCR not only as a research tool and a means for early diagnostic applications in clinical settings. The enhanced sensitivity of ddPCR could play a crucial role in the early detection of genetic markers associated with ovarian cancer, thereby facilitating timely therapeutic interventions.
Tumor multidrug resistance (MDR) is a growing public health concern, significantly impacting mortality and morbidity rates across various cancers. Identifying and targeting novel molecular mechanisms involved in drug resistance is crucial for advancing therapeutic strategies. WWP1, identified as a proto-oncogene and an E3 ubiquitin ligase [14], is known for its overexpression or amplification in several cancer types, including gastric, breast, liver, lung, and prostate cancers [15–19]. This protein primarily undermines the tumor suppressor role of PTEN through multi-ubiquitination, inhibiting PTEN's dimerization and membrane localization. This inhibition triggers the activation of the PI3K/AKT signaling pathway, a critical promoter of tumor MDR [20].
Additionally, experimental evidence suggests that WWP1 interacts directly with miR-452, influencing cellular processes related to migration and invasion, particularly in prostate cancer (PCa) cells [21]. Silencing or inhibiting WWP1 has shown potential in suppressing these malignant characteristics. Therefore, developing inhibitors that target WWP1 could serve as a transformative approach for reversing drug resistance in cancer, presenting a promising avenue for enhancing the efficacy of current therapeutic regimens and potentially improving clinical outcomes in cancer management.
Extrachromosomal DNA (cDNA) is prevalent in various human cancers, significantly enhancing oncogene expression through gene amplification and regulatory modifications. Kim et al. conducted a computational analysis of whole-genome sequencing data from 3,212 cancer patients, revealing a substantial enrichment of oncogenes on amplified cDNA [22]. Such amplifications are commonly associated with recurrent oncogenes on ecDNA and correlate with poor 5-year survival rates in cancer patients, indicating a strong link between ecDNA presence and increased tumor invasiveness. This amplification is notably widespread across different cancer types.
Another pivotal study highlighted the role of cDNA in developing cross-resistance, particularly through the amplification of the MYC oncogene. EcDNA, which includes MYC paralogs, contributes significantly to this resistance mechanism [23]. MYC's interaction with WWP1 is crucial, as it can directly induce multidrug resistance (MDR) in tumors. This interaction underlines the rationale behind developing a digital PCR (ddPCR) technique to evaluate how treatment regimens affect WWP1-eccDNA levels.
Hydroxyurea (HU), a cell cycle-specific chemotherapeutic agent that inhibits nucleoside diphosphate reductase, effectively blocks DNA synthesis during the S phase, thus curbing tumor cell proliferation [24]. Research has shown that hydroxyurea can also reduce tumor heterogeneity by eliminating ecDNA-containing MYC amplifications [25]. Since WWP1 is a downstream gene of MYC, inhibiting WWP1-eccDNA by hydroxyurea could be a potential mechanism for reversing tumor resistance.
Our comparative analysis of WWP1-eccDNA levels across different administration methods revealed that while cisplatin treatment increases WWP1-eccDNA content, hydroxyurea treatment decreases it. These results affirm the differential impact of these drugs on eccDNA dynamics and, by extension, on tumor chemoresistance.
While innovative, the research methodology implemented in this study is not without limitations. Digital droplet PCR (ddPCR), though it offers enhanced sensitivity and specificity over quantitative PCR (qPCR), also comes with higher consumable costs and generally detects a lower quantity of nucleic acids per test. These constraints highlight areas for potential improvement in the efficiency and cost-effectiveness of ddPCR technology.
Despite these limitations, the ddPCR method developed here holds considerable promise for broad applications in cancer research. It provides a more precise quantification of extrachromosomal circular DNA (eccDNA) levels within cells, which can be crucial for understanding the molecular dynamics in various types of tumors. This method has significant implications for detecting eccDNA across different cancer domains, enhancing our ability to study its role in oncogenesis and resistance mechanisms.
In conclusion, WWP1 plays a pivotal role in developing tumor resistance, primarily through its involvement in the formation and function of eccDNA. It is highly expressed in ovarian cancer cells, contributing significantly to their resistance profiles. These findings offer valuable insights into potential targets for novel therapeutic approaches to treat ovarian cancer and overcome drug resistance. This research sheds light on the biological underpinnings of chemoresistance and sets the stage for future clinical strategies that could revolutionize the management of this challenging disease.