Supplementary Figure S3. (a) Representative images of carmine staining of whole mount FVB normal mammary gland compared to vehicle- andHC4-treated MMTV-HER2 mammary gland whole mount. Scale bar 2 mm.(b) Examples of the mammary gland histological structures quantified.Scale bar 3 mm. Number of normal branches in the mammary gland ductal tree of non-tumor bearing healthy mice treated with vehicle (N=14) orHC4 (N=10). P by Mann-Whitney test. (c) The panels show representative images of histological sections showing at low magnification (top row 100µm scale bar) the indicated ductal and early lesion structures; the lower row showed higher magnification (25 µm scale bars) these structures. Theseimages were used as a key to classify per animal the effects of control or HC4 treatments.
ARTICLE ABSTRACT
The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis.
A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR.
HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF.
Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested “persister” cells that escape anti-proliferative therapies.
