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Supplementary Data from Hypoxic Activation of the PERK/eIF2α Arm of the Unfolded Protein Response Promotes Metastasis through Induction of LAMP3

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posted on 2023-03-31, 17:40 authored by Hilda Mujcic, Anika Nagelkerke, Kasper M.A. Rouschop, Stephen Chung, Naz Chaudary, Paul N. Span, Blaise Clarke, Michael Milosevic, Jenna Sykes, Richard P. Hill, Marianne Koritzinsky, Bradly G. Wouters

Supplementary Data - PDF file 113K, This file contains supplementary materials and methods, and the legends of the 14 supplementary figures. Suppl. Table 1 shows the patients characteristic from the clinical study described in main Fig 1. Suppl. Table 2 shows the relationship between LAMP3 protein expression and continuous clinical parameters, incl. HP5. Suppl. Table 3 shows the relationship between LAMP3 protein expression and categorical clinical parameters, incl. FIGO stage. Suppl. Fig 1 shows the association between LAMP3 gene copy number and LAMP3 gene expression in human cervix tumors described in main Fig 1, as well as the LAMP3 gene copy numbers in normal human cervix tissues. Suppl. Fig 2 provides in vitro evidence for the inducibility of the ME180 cancer cell line. Suppl. Fig 3 provides in vivo evidence for the metastatic capacity of the isogenic ME180 cell line, as well as its inducibility. Suppl. Fig 4 shows in vitro validation of the ME180-GADD34-C cells. Suppl. Fig 5 contains the raw data that belongs to main Fig 3C, which shows that Inhibition of UPR signaling suppresses hypoxia-induced lymph node metastasis. Suppl. Fig 6 shows the in vitro validation of ME180 LAMP3-knockdown cells. Suppl. Fig 7 shows representative images of LAMP3 expression in LAMP3 knockdown and empty vector control tumors described in main Fig 4B. Suppl. Fig 8 contains the raw data that belongs to main Fig 4C, which shows that knockdown of LAMP3 inhibits hypoxia-induced lymph node metastasis. Suppl. Fig 9 shows the human VEGF mRNA expression in ME180-GADD34-C orthotopic xenografts described in main Fig 3B. Suppl. Fig 10 shows the quantification of the microvessel density and hVEGF expression in shLAMP3 orthotopic xenografts described in main Fig 4B. Suppl. Fig 11 shows the hypoxic fraction shLAMP3 orthotopic xenografts described in main Fig 4B. Suppl. Fig 12 contains data from the soft agar colony formation assay that shows that knockdown of LAMP3 in HeLa cells inhibits colony formation. Suppl. Fig 13 is supplementary to main Fig 6B and shows that knockdown of LAMP3 in HeLa cells, just like in ME180 cells, inhibits transwell migration. Suppl. Fig 14 is supplementary to main Fig 6D and shows that knockdown of LAMP3 in Caski cells, just like in ME180 cells, inhibits wound closure in the scratch wound healing assay. This figure also shows that overexpression of LAMP3 accelerates wound closure

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ARTICLE ABSTRACT

Purpose: Conditions of poor oxygenation (hypoxia) are present in many human tumors, including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated.Experimental Design: Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis.Results: We show that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due, in part, to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration.Conclusion: These data suggest that the association between hypoxia, metastasis, and poor prognosis is due, in part, to hypoxic activation of the UPR and expression of LAMP3. Clin Cancer Res; 19(22); 6126–37. ©2013 AACR.