PERK Is a Haploinsufficient Tumor Suppressor: Gene Dose Determines Tumor-Suppressive Versus Tumor Promoting Properties of PERK in Melanoma

Abstract

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK.

Fig 1. Deletion of one Perk allele cooperates with Braf^V600E to drive metastatic melanoma.

A) Kaplan-Mayer survival curve of TyrCre+/-;Braf^V600ECA/+;Perk+/- mice treated with 4-HT. B) Western blot of melanoma skin lysates from TyrCre+/-;Braf^V600ECA/+;Fbxo4;Perk +/+ or TyrCre+/-;Braf^V600ECA/+;Perk+/- or Perk-/- mice (blot anti-Perk, p-eIF2α, eIF2α). C) Melanoma from TyrCre+/-;Braf^V600ECA/+;Perk+/- mice analyzed by H&E. Scale bars = 50μm.

Fig 2. Mono-allelic Perk deletion reduces pro-apoptotic signals and maintains pro-survival signaling.

H&E and IHC of premalignant skin from TyrCreCA/+;Braf^V600ECA/+, Perk +/+ or Perk+/- and Perk-/- mice for the indicated targets and quantification of staining/staining index (SI) of IHC; scale bars = 50μm.

Fig 3. Braf^V600E induces ER stress and reduced PERK gene dosage attenuates Braf^V600E-dependent senescence in premalignant skin.

A) Braf^V600E-dependent regulation of ER stress genes (CHOP, Grp78, Grp94, ATF4, GADD34, ERDJ4, XBP1) in primary melanocytes (QPCR; *p<0.05; p-values analyzed by two-tailed Student t test) B) β-galactosidase staining of primary Braf^WT vs Braf^V600E melanocytes. (Scale bars 50μm; *p<0.05; p-values analyzed by two-tailed Student t test); western analysis of cell lysates using indicated antibodies. C) UPR gene expression assay (QPCR) for Chop, Xbp1, Bip in premalignant skin from Braf^WT or Braf^V600E mice. D) β-galactosidase assay in premalignant skin isolated from TyrCreCA/+; Braf^V600ECA/+; Perk+/+ or Perk+/- or Perk-/- mice. Arrowheads indicate positive staining. Scale bars = 50μm. E) Detection of cyclin D1 in the tissues indicated by immunoblot. Total Erk1/2 is provided as a loading control. The line denotes excision of irrelevant lanes. F) Quantification of E. Cyclin D1 protein levels expressed as a ratio relative with total ERK.

Fig 4. Braf^V600ECA/+;Perk+/- tumors are dependent upon the remaining Perk allele.

A) LY4 PERK inhibitor inhibits stress-dependent PERK signaling in cultured melanoma cells. B-C) Measurement of melanoma tumor volume B) and tumor weight C) in TyrCre+/-;Braf^V600ECA/+;Perk +/- mice treated with LY-4; p-values analyzed by two-tailed Student t test. D-F) Western blot of melanoma skin lysates obtained from TyrCre+/-;Braf^V600ECA/+;Perk +/- mice treated with PERK inhibitor LY-4. G) Kaplan-Mayer survival curve of TyrCre+/-;Braf^V600ECA/+;Chop+/- or TyrCre+/-;Braf^V600ECA/+;Chop-/- mice expressed relative to control genotypes. H) Kaplan-Mayer survival curve for Braf^V600ECA/+;D1+/-;Perk+/- expressed relative to control genotypes.

Fig 5. Bi-allelic Perk deletion abolishes Braf^V600ECA/+;Fbxo4+/- melanoma initiation, but Perk inhibition cannot recede tumor progression.

A) Kaplan-Mayer survival curve of Braf^V600ECA/+, Fbxo4 +/- and Perk +/+, +/- or -/- mice treated with 4-Hydroxytamoxifen (4-HT). B) Western blot of melanoma lysates from TyrCreCA/+; Braf^V600ECA/+; Fbxo4 +/-; Perk +/+, Perk+/- or Perk-/- mice C) IHC analysis of skin from TyrCre+/-;Braf^V600ECA/+;Fbxo4 +/-; Perk+/+ or Perk -/- mice; H&E or IHC with antibodies for S100, p-eIF2α, Chop, cyclin D1, p-Akt, CD31 and quantification of staining/staining index (SI) of IHC. Scale bars = 50μm. D) Tumor volume in TyrCre+/-;Braf^V600ECA/+;Pten-/- mice treated with LY-4; p-values analyzed by two-tailed Student t test. E-F) Western blot of melanoma skin lysates from TyrCre+/-; Braf^V600ECA/+;Pten-/- mice treated with LY-4.

Fig 6. PERK is active in human melanoma cell lines and is required for melanoma genesis.

A) PERK is functional human melanoma cell lines. B) Dox-dependent PERK knockdown in WM3918 cells. C) WM3918 and WM239A growth in soft agar with or without PERK knockdown, +/- thapsigargin (Tg); V-vector; doxycycline (Dox); shPERK (soft agar); p-values analyzed by two-tailed Student t test D) Clonogenic survival assay of PERK knockdown WM3918 cells treated with thapsigargin (Tg); doxycycline. E) Cell cycle profile of PERK knockdown WM3918 cells.

Fig 7. Characterization of a tumor-derived mutation of the PERK kinase.

A) Depection of melanoma-derived PERK mutations. B-D) Western analysis of MEF lysates expressing murine versions of Perk alleles tested under ER-stress conditions. E) Clonogenic survival assay of Perk-/- fibroblasts reconstituted with Perk alleles (indicated on left) and treated with with chemicals indicated at the top. Cells stained with Geimsa after 14 days post-treatmen. F) Quantification of colonies that formed in soft agar for Perk-/- fibroblasts reconsituted with the indicated Perk mutants. G) Tumor-derived Perk alleles exhibit dominant negative activity.