Drug-Resistant Epithelial Ovarian Cancer: Current and Future Perspectives.

Epithelial ovarian cancer (EOC) is a complex disease with diverse histological subtypes, which, based on the aggressiveness and course of disease progression, have recently been broadly grouped into type I (low-grade serous, endometrioid, clear cell, and mucinous) and type II (high-grade serous, high-grade endometrioid, and undifferentiated carcinomas) categories. Despite substantial differences in pathogenesis, genetics, prognosis, and treatment response, clinical diagnosis and management of EOC remain similar across the subtypes. Debulking surgery combined with platinum-taxol-based chemotherapy serves as the initial treatment for High Grade Serous Ovarian Carcinoma (HGSOC), the most prevalent one, and for other subtypes, but most patients exhibit intrinsic or acquired resistance and recur in short duration. Targeted therapies, such as anti-angiogenics (e.g., bevacizumab) and PARP inhibitors (for BRCA-mutated cancers), offer some success, but therapy resistance, through various mechanisms, poses a significant challenge. This comprehensive chapter delves into emerging strategies to address these challenges, highlighting factors like aberrant miRNAs, metabolism, apoptosis evasion, cancer stem cells, and autophagy, which play pivotal roles in mediating resistance and disease relapse in EOC. Beyond standard treatments, the focus of this study extends to alternate targeted agents, including immunotherapies like checkpoint inhibitors, CAR T cells, and vaccines, as well as inhibitors targeting key oncogenic pathways in EOC. Additionally, this chapter covers disease classification, diagnosis, resistance pathways, standard treatments, and clinical data on various emerging approaches, and advocates for a nuanced and personalized approach tailored to individual subtypes and resistance mechanisms, aiming to enhance therapeutic outcomes across the spectrum of EOC subtypes.

Correction: Mukherjee et al. Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway. Cancers 2022, 14, 3365.

In the original publication [...].

Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase-Luciferin Pair.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.

Through the Looking Glass: Updated Insights on Ovarian Cancer Diagnostics.

Epithelial ovarian cancer (EOC) is the deadliest gynaecological malignancy and the eighth most prevalent cancer in women, with an abysmal mortality rate of two million worldwide. The existence of multiple overlapping symptoms with other gastrointestinal, genitourinary, and gynaecological maladies often leads to late-stage diagnosis and extensive extra-ovarian metastasis. Due to the absence of any clear early-stage symptoms, current tools only aid in the diagnosis of advanced-stage patients, wherein the 5-year survival plummets further to less than 30%. Therefore, there is a dire need for the identification of novel approaches that not only allow early diagnosis of the disease but also have a greater prognostic value. Toward this, biomarkers provide a gamut of powerful and dynamic tools to allow the identification of a spectrum of different malignancies. Both serum cancer antigen 125 (CA-125) and human epididymis 4 (HE4) are currently being used in clinics not only for EOC but also peritoneal and GI tract cancers. Screening of multiple biomarkers is gradually emerging as a beneficial strategy for early-stage diagnosis, proving instrumental in administration of first-line chemotherapy. These novel biomarkers seem to exhibit an enhanced potential as a diagnostic tool. This review summarizes existing knowledge of the ever-growing field of biomarker identification along with potential future ones, especially for ovarian cancer.

Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway.

An active fluidic microenvironment governs peritoneal metastasis in epithelial ovarian cancer (EOC), but its critical functional/molecular cues are not fully understood. Utilizing co-culture models of NIH3T3 cells (differentially overexpressing Jagged1) and SKOV3 cells expressing a Notch3 luciferase reporter-sensor (SNFT), we showed that incremental expression of Jagged1 led to proportional Notch3 activation in SNFT. With no basal luciferase activity, this system efficiently recorded dose-dependent Notch3 activation by rh-Jag1 peptide and the non-appearance of such induction in co-culture with NIH3T3Δjag1 cells indicates its sensitivity and specificity. Similar Notch3 modulation was shown for the first time in co-cultures with HGSOC patients' ascites-derived cancer-associated fibroblasts and Jagged1-expressing EOC cell lines. NIH3T3J1-A and OVCAR3 co-cultured SNFT cells showed maximum proliferation, invasion, and cisplatin resistance among all the heterotypic/homotypic cellular partners. VEGFA and CDKN1A are the two most upregulated genes identified across co-cultures by the gene profiler array. Co-culture induced VEGFA secretion from SNFT cells which also reduced cancer stem cell differentiation in platinum-resistant A2780 cells. rh-Jag1-peptide promoted enhanced nuclear-cytoplasmic p21 expression. Additionally, metastatic HGSOC tumors had higher VEGFA than corresponding primary tumors. This study thus demonstrates the tumoral and non-tumoral cell-mediated differential Notch3 activation imparting its tumorigenic effects through two critical molecular regulators, VEGFA and p21, during EOC progression.

Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance.

Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin-paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome-lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum-taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum-taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.