Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and currently lacks any effective targeted therapy. Since epigenetic alterations are a common event in TNBC, DNA methylation profiling can be useful for identifying potential biomarkers and therapeutic targets. Here, genome-wide DNA methylation from eight TNBC and six non-neoplastic tissues was analysed using Illumina Human Methylation 450K BeadChip. Results were validated by pyrosequencing in an independent cohort of 50 TNBC and 24 non-neoplastic samples, where protein expression was also assessed by immunohistochemistry. The functional role of disintegrin and metalloproteinase domain-containing protein 12(ADAM12) in TNBC cell proliferation, migration and drug response was analysed by gene expression silencing with short hairpin RNA. Three genes (Von Willenbrand factor C and Epidermal Growth Factor domain-containing protein (VWCE), tetraspanin-9 (TSPAN9) and ADAM12) were found to be exclusively hypomethylated in TNBC. Furthermore, ADAM12 hypomethylation was associated with a worse outcome in TNBC tissues and was also found in adjacent-to-tumour tissue and, preliminarily, in plasma from TNBC patients. In addition, ADAM12 silencing decreased TNBC cell proliferation and migration and improved doxorubicin sensitivity in TNBC cells. Our results indicate that ADAM12 is a potential therapeutic target and its hypomethylation could be a poor outcome biomarker in TNBC.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix –SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell^® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype and lacks targeted treatment. It is diagnosed by the absence of immunohistochemical expression of several biomarkers, but this method still displays some interlaboratory variability. DNA methylome aberrations are common in BC, thereby methylation profiling could provide the identification of accurate TNBC diagnosis biomarkers. Here, we generated a signature of differentially methylated probes with class prediction ability between 5 non-neoplastic breast and 7 TNBC tissues (error rate = 0.083). The robustness of this signature was corroborated in larger cohorts of additional 58 non-neoplastic breast, 93 TNBC, and 150 BC samples from the Gene Expression Omnibus repository, where it yielded an error rate of 0.006. Furthermore, we validated by pyrosequencing the hypomethylation of three out of 34 selected probes (FLJ43663, PBX Homeobox 1 (PBX1), and RAS P21 protein activator 3 (RASA3) in 51 TNBC, even at early stages of the disease. Finally, we found significantly lower methylation levels of FLJ43663 in cell free-DNA from the plasma of six TNBC patients than in 15 healthy donors. In conclusion, we report a novel DNA methylation signature with potential predictive value for TNBC diagnosis.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.