RESUMO
Plasma cell-free DNA (cfDNA) is a promising source of gene mutations for cancer detection by liquid biopsy. However, no current tests interrogate chromosomal structural variants (SVs) genome-wide. Here, we report a simple molecular and sequencing workflow called Genome-wide Analysis of Palindrome Formation (GAPF-seq) to probe DNA palindromes, a type of SV that often demarcates gene amplification. With low-throughput next-generation sequencing and automated machine learning, tumor DNA showed skewed chromosomal distributions of high-coverage 1-kb bins (HCBs), which differentiated 39 breast tumors from matched normal DNA with an average Area Under the Curve (AUC) of 0.9819. A proof-of-concept liquid biopsy study using cfDNA from prostate cancer patients and healthy individuals yielded an average AUC of 0.965. HCBs on the X chromosome emerged as a determinant feature and were associated with androgen receptor gene amplification. As a novel agnostic liquid biopsy approach, GAPF-seq could fill the technological gap offering unique cancer-specific SV profiles.
RESUMO
A major metastasis suppressing mechanism is the rapid apoptotic death of cancer cells upon detachment from extracellular matrix, a process called anoikis. Focal adhesion kinase (PTK2/FAK) is a key enzyme involved in evasion of anoikis. We show that loss of the Cub-domain containing protein-1 (CDCP1), paradoxically stimulates FAK activation in the detached state of prostate cancer cells. In CDCP1low DU145 and PC3 prostate cancer cells, detachment-activation of FAK occurs through local production of PI(4,5)P2. PI(4,5)P2 is generated by the PIP5K1c-201 splicing isoform of PIP5K1c, which contains a unique SRC phosphorylation site. In the detached state, reduced expression of CDCP1 and an alternative CDCP1-independent SRC activation mechanism triggers PIP5K1c-pY644 phosphorylation by SRC. This causes a switch of Talin binding from ß1-integrin to PIP5K1c-pY644 and leads to activation of PIP5K1c-FAK. Reduced CDCP1 expression also inactivates CDK5, a negative regulator of PIP5K1c. Furthermore, immersion of prostate cancer cells in 10% human plasma or fetal bovine serum is required for activation of PIP5K1c-FAK. The PIP5K1c induced detachment-activation of FAK in preclinical models sensitizes CDCP1low prostate cancer cells to FAK inhibitors. In patients, CDCP1High versus CDCP1low circulating tumor cells differ in expression of AR-v7, ONECUT2 and HOXB13 oncogenes and TMPRSS2 and display intra-patient heterogeneity of FAK-pY397 expression. Taken together, CDCP1low and CDCP1high detached prostate cancer cells activate distinct cytoplasmic kinase complexes and targetable transcription factors, which has important therapeutic implications.