Inhibition of eIF2alpha dephosphorylation inhibits ErbB2-induced deregulation of mammary acinar morphogenesis.
BACKGROUND
The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in approximately 30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2alpha inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2alpha can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2alpha phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.


RESULTS
We tested whether ErbB2 signaling influenced eIF2alpha signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt) Neu/ErbB2 or a constitutively active (CA) variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2alpha signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2alpha or induction of its downstream target ATF4. However, inhibition of eIF2alpha dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells.


CONCLUSIONS
Depending on the strength of ErbB2 signaling there is a differential regulation of CHOP and eIF2alpha phosphorylation. ErbB2 uncouples in basal conditions eIF2alpha phosphorylation from CHOP induction. However, this signal was restored by salubrinal treatment in Wt-ErbB2 expressing MCF10A cells as these DCIS-like structures underwent luminal clearing. In CA-ErbB2 structures apoptosis is not induced by salubrinal and instead a state of quiescence with reduced proliferation was achieved. Treatments that stabilize P-eIF2alpha levels may be effective in treating ErbB2 positive cancers without severely disrupting normal tissue function and structure.
Sequeira
Sharon J
SJ
Department of Medicine, Division of Hematology and Oncology, Tisch Cancer Institute at Mount Sinai, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. ss7267@albany.edu
Wen
Huei Chi
HC
Avivar-Valderas
Alvaro
A
Farias
Eduardo F
EF
Aguirre-Ghiso
Julio A
JA
D051701:Activating Transcription Factor 4 / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D051379:Mice; D009024:Morphogenesis; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000378:metabolism
D051701:Activating Transcription Factor 4; D019938:Cyclin D1; D015852:Eukaryotic Initiation Factor-2; D018719:Erb-b2 Receptor Tyrosine Kinases; D051742:Transcription Factor CHOP; C081452:Atf4 protein, mouse; C530105:Ccnd1 protein, mouse; C497249:DDIT3 protein, human