Tissue mechanics promote IDH1-dependent HIF1α–tenascin C feedback to regulate glioblastoma aggression
Authors: Khadjia Lobo
Joanna J Phillips
Michael W Pickup
Yekaterina A Miroshnikova
Johnathan N Lakins
J Matthew Barnes
Valerie M Weaver
Gerald F Reis
Tracy R McKnight
Janna K Mouw
Eric C Holland
Youngmi Kim
Anders I Persson
Journal: Nature Cell Biology (2016)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: glioma; IDH1; HIF1α; ECM; TNC
Affiliations: Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California San Francisco, San Francisco, California 94143, USA., Division of Human Biology and Solid Tumor Translational Research, Fred Hutchinson Cancer Research Center, Department of Neurosurgery and Alvord Brain Tumor Center, University of Washington, Seattle, Washington 98109, USA., Magnetic Resonance Science Center, Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, California 94143, USA., Department of Neurology, University of California, San Francisco, California 94143, USA., Department of Neurological Surgery, University of California San Francisco, San Francisco, California 94158, USA., Brain Tumor Research Center, Helen Diller Family Cancer Research Center, University of California San Francisco, San Francisco, California 94143, USA., UCSF Comprehensive Cancer Center, Helen Diller Family Cancer Research Center, University of California San Francisco, San Francisco, California 94143, USA., Department of Pathology, University of California, San Francisco, California 94143, USA., Department of Anatomy and Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94143, USA., Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California 94143, USA., UCSF Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California 94143, USA.

Abstract:  Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status. 

Retracted: No

PubPeer comments:
null, 
null, 
 For any given biological replicate, multiple western blots were run in parallel to be able to 1) avoid stripping and re-probing (and any crossover of signal that might occur when using primaries of the same species one after another), and 2) use 1 10th of the protein lysate input for housekeeping purposes (and keep all probed targets within a linear and quantifiable range). This process was repeated for a minimum of three independent biological replicates. Only data from a single biological replicate (experiment) is presented; all biological replicates are quantified for graphical purposes. And, yes, the authors feel comfortable using parallel western blots from the SAME experiment; minimally, housekeeping genes often require less protein input to actually have any (semi)quantitative accuracy. , 
null, 
 I think the previous commenters are correct in their criticisms of this work. Regardless of whether or not the authors feel comfortable using this approach, it is simply inappropriate to run parallel blots with one blot being probed for the protein of interest and the other membrane for the *loading* control. The appropriate approach would be to standardize membrane stripping procedures in your laboratory so that one blot can be probed multiple times, independent of whether the same species antibody was previously used.. Loading control antibodies can be titrated to get an appropriate signal. , 
 #5 I appreciate the perspective but think - in this scenario - we are going to have to agree to disagree on this point.u00a0 1. While the suggestion to titer down the antibody concentration for the loading control is often appropriate, it doesn t change the fact that the protein itself (in this case - actin) is no longer in a linear range for quantification. Regardless of antibody concentration, there is not a linear relationship between the band intensity and the amount of target on the membrane (when the amount of that target is in such abundance). Signal saturation versus membrane saturation. No amount of reducing the antibody concentration can change the physics of the technique. 2. The amount of loaded protein necessary for assessing some proteins (such as tenascin C in this experiment), as well as the transfer conditions optimal for TNC in this series of experiments, often yields one contiguous actin band at 42kDa similar to a Groucho Marx mustache (even when running a 4-20% gradient gel). How do we know this? Because we tried.u00a0 While I understand the insistence in maintaining the loading on the same membrane as other targets, in reality, it doesn t always yield an accurate assessment. In the future, we can absolutely probe the same blot for the loading control, and present that data in the supplement (in addition to the 1 10 loading blot). Or, better yet (but not always possible), find a loading control that isn t expressed in such excess compared to the other proteins of interest. ,