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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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Migration of breast cancer cells correlates with LAMP3 mRNA expression. (A) Migration of six different breast cancer cell lines under normoxic conditions during 24 hours in a transwell assay. (B) mRNA expression of LAMP3 in the same six cell lines. Results are from two representative experiments with three replicates each. LAMP3, lysosomal-associated membrane protein 3.
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Figure 1: Migration of breast cancer cells correlates with LAMP3 mRNA expression. (A) Migration of six different breast cancer cell lines under normoxic conditions during 24 hours in a transwell assay. (B) mRNA expression of LAMP3 in the same six cell lines. Results are from two representative experiments with three replicates each. LAMP3, lysosomal-associated membrane protein 3.

Mentions: To study the role of the UPR in the process of breast cancer cell migration, a number of models for in vitro metastasis were used. First, cell migration was studied in a series of six breast cancer cell lines under normoxic conditions using a transwell assay. In this assay cells have to actively migrate through a porous membrane. Cells were serum-deprived overnight and added to the upper compartment of modified Boyden chambers. In the lower compartment serum was added as a chemoattractant. As shown in Figure 1A, vast differences were found in the capability of cells to migrate to the lower compartment. The largest percentage of migrated cells was found in MDA-MB-231 and MDA-MB-468 cells, both triple-negative cell lines (that is, lacking expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2). In contrast, MCF-7 and MDA-MB-175 cells demonstrated very little migration. A direct comparison between the potential to migrate and the expression of LAMP3 on the mRNA level revealed a moderate correlation in this small group of cell lines (see Figure 1B). All subsequent experiments were performed with the MDA-MB-231 cells, a commonly used breast cancer cell line to study cell migration.


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)

Migration of breast cancer cells correlates with LAMP3 mRNA expression. (A) Migration of six different breast cancer cell lines under normoxic conditions during 24 hours in a transwell assay. (B) mRNA expression of LAMP3 in the same six cell lines. Results are from two representative experiments with three replicates each. LAMP3, lysosomal-associated membrane protein 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Migration of breast cancer cells correlates with LAMP3 mRNA expression. (A) Migration of six different breast cancer cell lines under normoxic conditions during 24 hours in a transwell assay. (B) mRNA expression of LAMP3 in the same six cell lines. Results are from two representative experiments with three replicates each. LAMP3, lysosomal-associated membrane protein 3.
Mentions: To study the role of the UPR in the process of breast cancer cell migration, a number of models for in vitro metastasis were used. First, cell migration was studied in a series of six breast cancer cell lines under normoxic conditions using a transwell assay. In this assay cells have to actively migrate through a porous membrane. Cells were serum-deprived overnight and added to the upper compartment of modified Boyden chambers. In the lower compartment serum was added as a chemoattractant. As shown in Figure 1A, vast differences were found in the capability of cells to migrate to the lower compartment. The largest percentage of migrated cells was found in MDA-MB-231 and MDA-MB-468 cells, both triple-negative cell lines (that is, lacking expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2). In contrast, MCF-7 and MDA-MB-175 cells demonstrated very little migration. A direct comparison between the potential to migrate and the expression of LAMP3 on the mRNA level revealed a moderate correlation in this small group of cell lines (see Figure 1B). All subsequent experiments were performed with the MDA-MB-231 cells, a commonly used breast cancer cell line to study cell migration.

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