Induction of circulating ABCB1 transcripts under platinum-based chemotherapy indicates poor prognosis and a bone micrometastatic phenotype in ovarian cancer patients.

The drug efflux transporter P-glycoprotein, encoded by the ABCB1 gene, promotes acquired chemoresistance. We explored the presence and clinical relevance of circulating cell-free ABCB1 transcripts (cfABCB1tx) in ovarian cancer patients (173 longitudinal serum samples from 79 cancer patients) using digital droplet PCR. cfABCB1tx were readily detectable at primary diagnosis (median 354 mRNA copies/20 µl serum), paralleled FIGO-stage and predicted surgical outcome (p = 0.023, p=0.022, respectively). Increased cfABCB1tx levels at primary diagnosis indicated poor PFS (HR = 2.329, 95%CI:1.374-3.947, p = 0.0017) and OS (HR = 2.074, 95%CI:1.194-3.601, p = 0.0096). cfABCB1tx induction under platinum-based chemotherapy was an independent predictor for poor OS (HR = 2.597, 95%CI: 1.218-5.538, p = 0.013) and paralelled a micrometastatic phenotype, shaped by the presence of disseminated tumor cells in the bone marrow. A strong correlation was observed between cfABCB1tx and circulating transcripts of the metastasis-inducer MACC1, which is the transcriptional activator of ABCB1. Combined assessment of cfABCB1tx and circulating cell-free MACC1 transcripts (cfMACC1tx) resulted in an improved prognostic prediction, with  the cfABCB1tx-high/cfMACC1tx-high phenotype bearing the highest risk for relapse and death. Conclusively, we provide proof of principle, that ABCB1 transcripts are readily traceable in the liquid-biopsy of ovarian cancer patients, advancing a new dimension for systemic monitoring of ABCB1/P-glycoprotein expression dynamics.

Urinary-based detection of MSL, HE4 and CA125 as an additional dimension for predictive and prognostic modelling in ovarian cancer.

Objectives: We have recently described a predictive/prognostic model for ovarian cancer, exploiting commonly available clinico-pathological parameters and the ovarian serum biomarkers mesothelin (MSL), human epididymis protein 4 (HE4) and cancer-antigen 125 (CA125). Considering urine as a prototype non-invasive sample, we investigated whether serum levels of these biomarkers are mirrored in urine and compared their clinical relevance in matched serum vs. urine samples. Methods: MSL, HE4 and CA125 were quantified in urinary (n=172) and matched serum samples (n=188) from ovarian cancer patients (n=192) using the Lumipulse® G chemiluminescent enzyme immunoassay (Fujirebio). Results: While absolute concentrations of MSL or CA125 were higher in serum than in matched urine samples, HE4 concentrations were considerably higher in urine than in serum. Nonetheless, the levels of all three biomarkers strongly correlated between matched serum vs. urine samples and were unrelated to BRCA1/2 mutational status. Consequently, prediction of surgical outcome or relapse/death by MSL, HE4 or CA125 was similarly efficient among urinary- vs. serum-based detection. HE4 provided the highest capacity to predict surgical outcome or relapse/death among both body fluids (urine: AUC=0.854; serum: AUC=0.750, respectively). All clinically relevant findings regarding the investigated urinary biomarkers were equally reproducible among raw vs. creatinine-normalized datasets, suggesting that normalization may have subordinate priority for urine-based analysis of these biomarkers. Conclusion: We report that the capacity of MSL, HE4 and CA125 to predict surgical outcome and relapse/death is equivalent between serum vs. urine-based detection. Urinary biomarkers, in particular HE4, may provide an additional dimension for prognostic modeling in ovarian cancer.

Epigenetic Targeting to Overcome Radioresistance in Head and Neck Cancer.

(1) Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treatment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) phenotype. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epigenetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. (2) Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes involved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensitizing and CSC-targeting potential. (3) Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 trimethylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. (4) Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy.

ALDH1A1 drives prostate cancer metastases and radioresistance by interplay with AR- and RAR-dependent transcription.

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro, in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations.

Establishment and Molecular Characterization of an In Vitro Model for PARPi-Resistant Ovarian Cancer.

Overcoming PARPi resistance is a high clinical priority. We established and characterized comparative in vitro models of acquired PARPi resistance, derived from either a BRCA1-proficient or BRCA1-deficient isogenic background by long-term exposure to olaparib. While parental cell lines already exhibited a certain level of intrinsic activity of multidrug resistance (MDR) proteins, resulting PARPi-resistant cells from both models further converted toward MDR. In both models, the PARPi-resistant phenotype was shaped by (i) cross-resistance to other PARPis (ii) impaired susceptibility toward the formation of DNA-platinum adducts upon exposure to cisplatin, which could be reverted by the drug efflux inhibitors verapamil or diphenhydramine, and (iii) reduced PARP-trapping activity. However, the signature and activity of ABC-transporter expression and the cross-resistance spectra to other chemotherapeutic drugs considerably diverged between the BRCA1-proficient vs. BRCA1-deficient models. Using dual-fluorescence co-culture experiments, we observed that PARPi-resistant cells had a competitive disadvantage over PARPi-sensitive cells in a drug-free medium. However, they rapidly gained clonal dominance under olaparib selection pressure, which could be mitigated by the MRP1 inhibitor MK-751. Conclusively, we present a well-characterized in vitro model, which could be instrumental in dissecting mechanisms of PARPi resistance from HR-proficient vs. HR-deficient background and in studying clonal dynamics of PARPi-resistant cells in response to experimental drugs, such as novel olaparib-sensitizers.