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Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response.

Nagelkerke A, Bussink J, Mujcic H, Wouters BG, Lehmann S, Sweep FC, Span PN - Breast Cancer Res. (2013)

Bottom Line: Furthermore, hypoxic tumors are associated with a poor prognosis.A direct correlation was found between cell migration and baseline LAMP3 expression.Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.

Methods: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.

Results: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.

Conclusions: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

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Hypoxia has a profound effect on cell migration in a gap closure assay. Shown is the percentage of the gap that is closed after 16 hours. Results are from two representative experiments with two replicates each. Asterisks indicate statistical significance compared to the normoxic control.
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Figure 4: Hypoxia has a profound effect on cell migration in a gap closure assay. Shown is the percentage of the gap that is closed after 16 hours. Results are from two representative experiments with two replicates each. Asterisks indicate statistical significance compared to the normoxic control.

Mentions: To confirm and extend the data from the transwell assay, similar experiments were performed using a gap closure assay, which does not require serum deprivation. First, the effect of exposure to hypoxia on the migration of MDA-MB-231 breast cancer cells was studied. Figure 4 shows the percentage of the gap that the cells filled in a 16-hour period. Compared to normoxic conditions, 1% O2 showed the largest percentage of gap closure. 0.5% O2 revealed a moderate but still significant effect. Exposure to even lower oxygen concentrations, that is 0.1% O2, did not reveal an increase in gap closure speed compared to normoxia.


Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response.

Nagelkerke A, Bussink J, Mujcic H, Wouters BG, Lehmann S, Sweep FC, Span PN - Breast Cancer Res. (2013)

Hypoxia has a profound effect on cell migration in a gap closure assay. Shown is the percentage of the gap that is closed after 16 hours. Results are from two representative experiments with two replicates each. Asterisks indicate statistical significance compared to the normoxic control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672809&req=5

Figure 4: Hypoxia has a profound effect on cell migration in a gap closure assay. Shown is the percentage of the gap that is closed after 16 hours. Results are from two representative experiments with two replicates each. Asterisks indicate statistical significance compared to the normoxic control.
Mentions: To confirm and extend the data from the transwell assay, similar experiments were performed using a gap closure assay, which does not require serum deprivation. First, the effect of exposure to hypoxia on the migration of MDA-MB-231 breast cancer cells was studied. Figure 4 shows the percentage of the gap that the cells filled in a 16-hour period. Compared to normoxic conditions, 1% O2 showed the largest percentage of gap closure. 0.5% O2 revealed a moderate but still significant effect. Exposure to even lower oxygen concentrations, that is 0.1% O2, did not reveal an increase in gap closure speed compared to normoxia.

Bottom Line: Furthermore, hypoxic tumors are associated with a poor prognosis.A direct correlation was found between cell migration and baseline LAMP3 expression.Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.

Methods: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.

Results: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.

Conclusions: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Show MeSH
Related in: MedlinePlus