Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab

Persistent mitochondrial hyperfusion promotes G2/M accumulation and caspase-dependent cell death.

Cancer cells have several hallmarks that define their neoplastic behavior. One is their unabated replicative potential that allows cells to continually proliferate, and thereby contribute to increasing tumor burden. The progression of a cell through the cell cycle is regulated by a series of checkpoints that ensures successful transmission of genetic information, as well as various cellular components, including organelles and protein complexes to the two resulting daughter cells. The mitochondrial reticulum undergoes coordinated changes in shape to correspond with specific stages of the cell cycle, the most dramatic being complete mitochondrial fragmentation prior to cytokinesis. To determine whether mitochondrial fission is a required step to ensure proper mitochondrial segregation into two daughter cells, we investigated the importance of mitochondrial dynamics to cell cycle progression. We found that mitochondrial hyperfusion promotes a defect in cell cycle progression characterized by an inability for cells to exit G2/M. Additionally, extended periods of persistent mitochondrial fusion led to robust caspase-dependent cell death. The cell death signals were coordinated through activation and cleavage of caspase-8, promoting a potent death response. These results demonstrate the importance of mitochondrial dynamics in cell cycle progression, and that inhibiting mitochondrial fission regulators may provide a therapeutic strategy to target the replicative potential of cancer cells

Mitochondrial Hyperfusion Promotes Cellular Accumulation in G2/M.

<p>(A) Relative Drp1 and Mff expression (mRNA) was measured by qRT-PCR (relative to HPRT, normalized to control siRNA). Bars represent standard error of replicate experiments. (B) Mitochondrial morphology of U2OS_mitoEYFP following siRNA transfection (48 hours) of mitochondrial fission regulators, Drp1 and Mff compared to control. Insets are 4x magnification of boxed regions. (C) Cell cycle distributions of cells following knockdown of Control, Drp1, or Mff (48 hours) by FACSCaliber analysis of DNA area of propidium iodide-stained cells. The black histogram represents the original cell cycle profile, while the shaded areas represent the fitted cell cycle model following analysis with Modfit LT. The histogram indicates the percentage of cells in G1 (first red peak positioned over 50), G2/M (second red peak positioned over 100), and S phase (the dashed area lying between the two red peaks). Aneuploidy populations are indicated by the green shaded area located along the axis at DNA areas 150 to 200. The percentage of cells in G2/M represents the average (± standard deviation) calculated from two independent experiments.</p

Mitochondrial Fragmentation Prior to Cytokinesis.

<p>(A) Mitochondria were tracked through mitotic division events in U2OS_mitoEYFP cells by confocal microscopy. An example cell is shown representing a 3D reconstruction of a 6 μm z-stack (1 μm thick slices) that has been re-colored according to the depth (see color coded legend) of the fluorescent signal within the z-stack. Insets are 3.5x magnifications of boxed regions. (B) Example cells are shown which demonstrate similar morphological alterations in mitochondrial reticulum throughout multiple mitotic cell divisions.</p

Mitochondrial Hyperfusion Promotes Caspase 8 Dependent Cell Death.

<p>(A-C) U2OS cells were transfected with control (black), Drp1 (green), or Mff (red) siRNA for 96 hours. Cells were collected and stained for (A) cleaved caspase 3 or (B) cleaved caspase 8. Positivity of cleaved caspase 3 or 8 is indicated by an increase in fluorescence on the FACS histogram compared to control. (C) U2OS cells transfected with control, Drp1, or Mff siRNAs were treated with pan caspase inhibitor, zVAD (20 μM; dark gray bars), or the necroptosis inhibitor, necrostatin (10 μM; light gray bars), or vehicle (DMSO; black bars) every 24 hours for 96 hours following transfection. Cells were stained with propidium iodide (PI) and percent PI positivity was used as a marker of cell death. Error bars represent standard deviation from two replicate experiments where at least 10,000 events were collected for each treatment.</p

Mitochondrial Hyperfusion Induces Cell Death in addition to Cell Cycle Delay.

<p>(A) Time-lapse images tracking U2OS_mitoEYFP for 18 hours following transfection of Drp1, Mff or control siRNAs. Individual cells were tracked throughout the time series to determine if they underwent mitotic cell division events (white arrows). (B) Graphical representation of the percentage of cells in each condition (at least 75 cells tracked for each condition) that underwent a mitotic cell division, underwent cell death (measured by cell retraction, nuclear condensation, and membrane blebbing) or underwent no event.</p

Quantitative analysis of tyrosine phosphorylation from FFPE tissues reveals patient-specific signaling networks

Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for in-depth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. In addition, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from one to two 10-μm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and tandem mass tag multiplexing enabled in-depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash-frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens. SIGNIFICANCE: This study reports a highly sensitive method utilizing FFPE tissues to identify dysregulated signaling networks in patient tumors, opening the door for direct translational insights from FFPE tumor tissue banks in hospitals