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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.

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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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Knockdown of LAMP3 reduces invasion of spheroids into collagen. (A) Expression of LAMP3 mRNA in control cells versus LAMP3 knockdown cells. (B) Spheroid size of MDA-MB-231 control and LAMP3 knockdown spheroids after 4 days of growth. Results are from two independent experiments with 32 replicates each. (C) F-actin staining of MDA-MB-231 spheroids of control and LAMP3 knockdown cells, after 6 days of invasion in collagen. Bar is 100 μm, original magnification is 50 x. (D) Surface of collagen invaded by MDA-MB-231 control and LAMP3 knockdown spheroids during 72 hours. (E) Collagen invasion of a mixed MDA-MB-231 spheroid after 6 days. Control cells were labeled with CellTracker Green and LAMP3 knockdown cells were labeled with CellTracker Orange. Control and LAMP3 knockdown cells were mixed in a 1:1 ratio. Bar is 100 μm, original magnification is 100 x. (F) Quantification of E. The total amount of green and red signal was analyzed in five different zones: the spheroid body and four consecutive invasive zones, n = 5. LAMP3, lysosomal-associated membrane protein 3.
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Figure 7: Knockdown of LAMP3 reduces invasion of spheroids into collagen. (A) Expression of LAMP3 mRNA in control cells versus LAMP3 knockdown cells. (B) Spheroid size of MDA-MB-231 control and LAMP3 knockdown spheroids after 4 days of growth. Results are from two independent experiments with 32 replicates each. (C) F-actin staining of MDA-MB-231 spheroids of control and LAMP3 knockdown cells, after 6 days of invasion in collagen. Bar is 100 μm, original magnification is 50 x. (D) Surface of collagen invaded by MDA-MB-231 control and LAMP3 knockdown spheroids during 72 hours. (E) Collagen invasion of a mixed MDA-MB-231 spheroid after 6 days. Control cells were labeled with CellTracker Green and LAMP3 knockdown cells were labeled with CellTracker Orange. Control and LAMP3 knockdown cells were mixed in a 1:1 ratio. Bar is 100 μm, original magnification is 100 x. (F) Quantification of E. The total amount of green and red signal was analyzed in five different zones: the spheroid body and four consecutive invasive zones, n = 5. LAMP3, lysosomal-associated membrane protein 3.

Mentions: Next, the ability of spheroids generated from cells with a stable knockdown of LAMP3 to invade a collagen matrix was studied. Figure 7A shows that stable knockdown of LAMP3 attenuated the mRNA expression of LAMP3. After 4 days of growth, both control and knockdown spheroids were of similar size, indicating that there is no difference in proliferation between these cells when grown as a spheroid (see Figure 7B). MDA-MB-231 negative control spheroids were found to invade the collagen with string-like protrusions (see Additional file 2). Stable knockdown of LAMP3 reduced these invasive filaments to some extent (see Figure 7C and Additional file 2). The effect of LAMP3 knockdown on invasion of the spheroids into collagen was quantified by measuring the surface of collagen invaded by the spheroids. The surface of the invasive zone was found to be smaller in the knockdown spheroids (see Figure 7D).


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)

Knockdown of LAMP3 reduces invasion of spheroids into collagen. (A) Expression of LAMP3 mRNA in control cells versus LAMP3 knockdown cells. (B) Spheroid size of MDA-MB-231 control and LAMP3 knockdown spheroids after 4 days of growth. Results are from two independent experiments with 32 replicates each. (C) F-actin staining of MDA-MB-231 spheroids of control and LAMP3 knockdown cells, after 6 days of invasion in collagen. Bar is 100 μm, original magnification is 50 x. (D) Surface of collagen invaded by MDA-MB-231 control and LAMP3 knockdown spheroids during 72 hours. (E) Collagen invasion of a mixed MDA-MB-231 spheroid after 6 days. Control cells were labeled with CellTracker Green and LAMP3 knockdown cells were labeled with CellTracker Orange. Control and LAMP3 knockdown cells were mixed in a 1:1 ratio. Bar is 100 μm, original magnification is 100 x. (F) Quantification of E. The total amount of green and red signal was analyzed in five different zones: the spheroid body and four consecutive invasive zones, n = 5. LAMP3, lysosomal-associated membrane protein 3.
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Related In: Results  -  Collection

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Figure 7: Knockdown of LAMP3 reduces invasion of spheroids into collagen. (A) Expression of LAMP3 mRNA in control cells versus LAMP3 knockdown cells. (B) Spheroid size of MDA-MB-231 control and LAMP3 knockdown spheroids after 4 days of growth. Results are from two independent experiments with 32 replicates each. (C) F-actin staining of MDA-MB-231 spheroids of control and LAMP3 knockdown cells, after 6 days of invasion in collagen. Bar is 100 μm, original magnification is 50 x. (D) Surface of collagen invaded by MDA-MB-231 control and LAMP3 knockdown spheroids during 72 hours. (E) Collagen invasion of a mixed MDA-MB-231 spheroid after 6 days. Control cells were labeled with CellTracker Green and LAMP3 knockdown cells were labeled with CellTracker Orange. Control and LAMP3 knockdown cells were mixed in a 1:1 ratio. Bar is 100 μm, original magnification is 100 x. (F) Quantification of E. The total amount of green and red signal was analyzed in five different zones: the spheroid body and four consecutive invasive zones, n = 5. LAMP3, lysosomal-associated membrane protein 3.
Mentions: Next, the ability of spheroids generated from cells with a stable knockdown of LAMP3 to invade a collagen matrix was studied. Figure 7A shows that stable knockdown of LAMP3 attenuated the mRNA expression of LAMP3. After 4 days of growth, both control and knockdown spheroids were of similar size, indicating that there is no difference in proliferation between these cells when grown as a spheroid (see Figure 7B). MDA-MB-231 negative control spheroids were found to invade the collagen with string-like protrusions (see Additional file 2). Stable knockdown of LAMP3 reduced these invasive filaments to some extent (see Figure 7C and Additional file 2). The effect of LAMP3 knockdown on invasion of the spheroids into collagen was quantified by measuring the surface of collagen invaded by the spheroids. The surface of the invasive zone was found to be smaller in the knockdown spheroids (see Figure 7D).

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