Pharmacological induction of selective endoplasmic reticulum retention as a strategy for cancer therapy

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository, with the data set identifier PXD014709. DAVID webserver was used for human database as background.
Unless mentioned otherwise, experiments were repeated at least three times. MTT and AST/ALT measurements were performed in triplicates and average was taken as a single data point. Average, standard deviation and p-values are shown for each experiment and calculated by two tailed student T-test. Immunoblotting was calculated by non-parametric tests owing to the non-linearity of this method. Animal studies were performed on cohorts at a size large enough to establish a statistical significane, as indicated in the methods.
No data were excluded All data was replicated in multiple cell types and for multiple sERr pharmacological combinations.
Animals were divided into treatment cohorts prior to tumor appearance to ensure a blind randomization.
The in vivo experiment was performed in a semi-blind fashion, in which technicians injected the mice and the analysis was done by students that are not familiar with the outcome.