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Hypoxia/HIF1α induces lapatinib resistance in ERBB2-positive breast cancer cells via regulation of DUSP2.

Karakashev SV, Reginato MJ - Oncotarget (2015)

Bottom Line: Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells.HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions.Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

ABSTRACT
ERBB2/HER2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can promote tumor progression. Breast cancer patients with ERBB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that specifically blocks EGFR/ERBB2 signaling. Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three-dimensional (3D) cultures are decreased under hypoxic conditions. Hypoxia can maintain activation of signaling pathways downstream from ERBB2 including AKT and ERK in the presence of lapatinib. HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions. Under hypoxia, activation of ERK signaling is required for lapatinib resistance as treatment with MEK inhibitor trametinib reverses hypoxia-mediated lapatinib resistance. HIF-1 can bypass the lapatinib-treated inhibition of the ERK pathway via inhibition of the dual-specificity phosphatase 2 (DUSP2). Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance. Thus, our results provide rationale for therapeutic evaluation of the treatment of hypoxic ERBB2 expressing breast tumors with a combination of lapatinib and MEK inhibitors.

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Hypoxia requires ERK activity for lapatinib resistance in breast cancer cells(A) Cells were treated with increasing doses of lapatinib under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. (B) Cells were treated with increasing doses of lapatinib under hypoxic conditions in the presence of control or trametinib (50 nM) and cell viability was assessed. (C) MCF10A-ERBB2 cells were placed in 3D culture conditions and then incubated under normoxic or hypoxic conditions in the presence or absence of lapatinib (1 μM), trametinib (50 nM) or both treatments. Cells were stained for cleaved caspase-3. (D) The percentage of caspase-positive acini was determined for C. (E) Cells were treated with lapatinib, trametinib or a combination of both drugs under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).
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Figure 2: Hypoxia requires ERK activity for lapatinib resistance in breast cancer cells(A) Cells were treated with increasing doses of lapatinib under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. (B) Cells were treated with increasing doses of lapatinib under hypoxic conditions in the presence of control or trametinib (50 nM) and cell viability was assessed. (C) MCF10A-ERBB2 cells were placed in 3D culture conditions and then incubated under normoxic or hypoxic conditions in the presence or absence of lapatinib (1 μM), trametinib (50 nM) or both treatments. Cells were stained for cleaved caspase-3. (D) The percentage of caspase-positive acini was determined for C. (E) Cells were treated with lapatinib, trametinib or a combination of both drugs under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).

Mentions: We next examined ERBB2 downstream signaling in lapatinib treated MCF10A-ERBB2 cells in response to hypoxia. As expected, lapatinib treatment of ERBB2-expressing cells reduced ERK and AKT activation under normoxic conditions (Figure 2A). However, under hypoxia ERK and AKT activation was increased and maintained even in presence of high doses of lapatinib (Figure 2A). Since recent studies have shown that hypoxia can also activate c-SRC [27], we examined c-SRC activation under these conditions. We found undetectable levels of c-SRC activation in normoxic cells. However, c-SRC is strongly activated in cells exposed to hypoxia and lapatinib treatment has minimal effects on c-SRC activation in these cells (Figure 2A). Thus, hypoxia increases and prolongs c-SRC, ERK and AKT activation in ERBB2-expressing cells in the presence of lapatinib.


Hypoxia/HIF1α induces lapatinib resistance in ERBB2-positive breast cancer cells via regulation of DUSP2.

Karakashev SV, Reginato MJ - Oncotarget (2015)

Hypoxia requires ERK activity for lapatinib resistance in breast cancer cells(A) Cells were treated with increasing doses of lapatinib under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. (B) Cells were treated with increasing doses of lapatinib under hypoxic conditions in the presence of control or trametinib (50 nM) and cell viability was assessed. (C) MCF10A-ERBB2 cells were placed in 3D culture conditions and then incubated under normoxic or hypoxic conditions in the presence or absence of lapatinib (1 μM), trametinib (50 nM) or both treatments. Cells were stained for cleaved caspase-3. (D) The percentage of caspase-positive acini was determined for C. (E) Cells were treated with lapatinib, trametinib or a combination of both drugs under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385829&req=5

Figure 2: Hypoxia requires ERK activity for lapatinib resistance in breast cancer cells(A) Cells were treated with increasing doses of lapatinib under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. (B) Cells were treated with increasing doses of lapatinib under hypoxic conditions in the presence of control or trametinib (50 nM) and cell viability was assessed. (C) MCF10A-ERBB2 cells were placed in 3D culture conditions and then incubated under normoxic or hypoxic conditions in the presence or absence of lapatinib (1 μM), trametinib (50 nM) or both treatments. Cells were stained for cleaved caspase-3. (D) The percentage of caspase-positive acini was determined for C. (E) Cells were treated with lapatinib, trametinib or a combination of both drugs under normoxic or hypoxic conditions and cell lysates were collected for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).
Mentions: We next examined ERBB2 downstream signaling in lapatinib treated MCF10A-ERBB2 cells in response to hypoxia. As expected, lapatinib treatment of ERBB2-expressing cells reduced ERK and AKT activation under normoxic conditions (Figure 2A). However, under hypoxia ERK and AKT activation was increased and maintained even in presence of high doses of lapatinib (Figure 2A). Since recent studies have shown that hypoxia can also activate c-SRC [27], we examined c-SRC activation under these conditions. We found undetectable levels of c-SRC activation in normoxic cells. However, c-SRC is strongly activated in cells exposed to hypoxia and lapatinib treatment has minimal effects on c-SRC activation in these cells (Figure 2A). Thus, hypoxia increases and prolongs c-SRC, ERK and AKT activation in ERBB2-expressing cells in the presence of lapatinib.

Bottom Line: Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells.HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions.Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

ABSTRACT
ERBB2/HER2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can promote tumor progression. Breast cancer patients with ERBB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that specifically blocks EGFR/ERBB2 signaling. Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three-dimensional (3D) cultures are decreased under hypoxic conditions. Hypoxia can maintain activation of signaling pathways downstream from ERBB2 including AKT and ERK in the presence of lapatinib. HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions. Under hypoxia, activation of ERK signaling is required for lapatinib resistance as treatment with MEK inhibitor trametinib reverses hypoxia-mediated lapatinib resistance. HIF-1 can bypass the lapatinib-treated inhibition of the ERK pathway via inhibition of the dual-specificity phosphatase 2 (DUSP2). Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance. Thus, our results provide rationale for therapeutic evaluation of the treatment of hypoxic ERBB2 expressing breast tumors with a combination of lapatinib and MEK inhibitors.

Show MeSH
Related in: MedlinePlus