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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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HIF-1 target DUSP2 is required for lapatinib resistance(A) Indicated cells were exposed to hypoxia for 6 hrs and cell lysates were collected for immunoblot analysis. (B) Cells stably expressing control or DUSP2 shRNA were treated with 1 μM lapatinib and cell lysates were collected for immunoblot analysis. (C) Cells expressing control or DUSP2 shRNA were treated with increasing doses of lapatinib and cell viability was assessed. (D) Cells stably expressing DUSP2-flag protein were treated with 1 μM lapatinib under hypoxia and cell lysates were collected for immunoblot analysis. (E) Cells expressing DUSP2-flag were treated with increasing doses of lapatinib and cell viability was assessed. (F) Cells as in E were stained with crystal violet. Error bars indicate S.E. (*p ≤ 0.05).
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Figure 5: HIF-1 target DUSP2 is required for lapatinib resistance(A) Indicated cells were exposed to hypoxia for 6 hrs and cell lysates were collected for immunoblot analysis. (B) Cells stably expressing control or DUSP2 shRNA were treated with 1 μM lapatinib and cell lysates were collected for immunoblot analysis. (C) Cells expressing control or DUSP2 shRNA were treated with increasing doses of lapatinib and cell viability was assessed. (D) Cells stably expressing DUSP2-flag protein were treated with 1 μM lapatinib under hypoxia and cell lysates were collected for immunoblot analysis. (E) Cells expressing DUSP2-flag were treated with increasing doses of lapatinib and cell viability was assessed. (F) Cells as in E were stained with crystal violet. Error bars indicate S.E. (*p ≤ 0.05).

Mentions: One possible mechanism of HIF1-mediated ERK activation under hypoxia is regulation of the dual specificity protein phosphatase 2 (DUSP2). DUSP2 is a phosphatase that negatively regulates ERK activity [31]. Recently, it has been reported that DUSP2 is downregulated in many cancers and that hypoxic tumors have decrease expression of DUSP2 [32]. We examined DUSP2 expression in three different ERBB2 expressing breast cancer cell lines and observed that DUSP2 protein levels are downregulated under hypoxic conditions (Figure 5A). DUSP2 RNA levels were also reduced nearly 90% in hypoxic MCF10-ERBB2 when compared to normal oxygen (Figure S5A). Consistent with its regulation of ERK, DUSP2 downregulation correlated with increased ERK activation in breast cancer cells placed under hypoxic conditions (Figure 5A). Moreover, MCF10A-ERBB2 cells expressing HIF-1α RNAi abrogated DUSP2 downregulation under hypoxia, (Figure S5B) thus regulation of DUSP2 by hypoxia in breast cancer cells is HIF-1α-dependent.


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

Karakashev SV, Reginato MJ - Oncotarget (2015)

HIF-1 target DUSP2 is required for lapatinib resistance(A) Indicated cells were exposed to hypoxia for 6 hrs and cell lysates were collected for immunoblot analysis. (B) Cells stably expressing control or DUSP2 shRNA were treated with 1 μM lapatinib and cell lysates were collected for immunoblot analysis. (C) Cells expressing control or DUSP2 shRNA were treated with increasing doses of lapatinib and cell viability was assessed. (D) Cells stably expressing DUSP2-flag protein were treated with 1 μM lapatinib under hypoxia and cell lysates were collected for immunoblot analysis. (E) Cells expressing DUSP2-flag were treated with increasing doses of lapatinib and cell viability was assessed. (F) Cells as in E were stained with crystal violet. 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 5: HIF-1 target DUSP2 is required for lapatinib resistance(A) Indicated cells were exposed to hypoxia for 6 hrs and cell lysates were collected for immunoblot analysis. (B) Cells stably expressing control or DUSP2 shRNA were treated with 1 μM lapatinib and cell lysates were collected for immunoblot analysis. (C) Cells expressing control or DUSP2 shRNA were treated with increasing doses of lapatinib and cell viability was assessed. (D) Cells stably expressing DUSP2-flag protein were treated with 1 μM lapatinib under hypoxia and cell lysates were collected for immunoblot analysis. (E) Cells expressing DUSP2-flag were treated with increasing doses of lapatinib and cell viability was assessed. (F) Cells as in E were stained with crystal violet. Error bars indicate S.E. (*p ≤ 0.05).
Mentions: One possible mechanism of HIF1-mediated ERK activation under hypoxia is regulation of the dual specificity protein phosphatase 2 (DUSP2). DUSP2 is a phosphatase that negatively regulates ERK activity [31]. Recently, it has been reported that DUSP2 is downregulated in many cancers and that hypoxic tumors have decrease expression of DUSP2 [32]. We examined DUSP2 expression in three different ERBB2 expressing breast cancer cell lines and observed that DUSP2 protein levels are downregulated under hypoxic conditions (Figure 5A). DUSP2 RNA levels were also reduced nearly 90% in hypoxic MCF10-ERBB2 when compared to normal oxygen (Figure S5A). Consistent with its regulation of ERK, DUSP2 downregulation correlated with increased ERK activation in breast cancer cells placed under hypoxic conditions (Figure 5A). Moreover, MCF10A-ERBB2 cells expressing HIF-1α RNAi abrogated DUSP2 downregulation under hypoxia, (Figure S5B) thus regulation of DUSP2 by hypoxia in breast cancer cells is HIF-1α-dependent.

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