Limits...
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.

Show MeSH

Related in: MedlinePlus

HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385829&req=5

Figure 4: HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).

Mentions: To determine whether HIF-1α stabilization is sufficient to confer lapatinib resistance in ERBB2-expressing cells under normoxic conditions, we overexpressed a stable non-degradable form of HIF-1α containing proline to alanine mutations (HIF-1α P402A, P564A) in MCF10A-ERBB2 cells. We confirmed that this mutant expressed levels similar to endogenous HIF-1α stabilized under hypoxia and to cells treated with prolyl-hydroxylase inhibitor DMOG (Figure 4A). MCF10A-ERBB2 cells expressing the stable form of HIF-1α were resistant to lapatinib-mediated effects on cell viability (Figure 4B) and cell number (Figure 4C) under normal oxygen conditions. In addition, similar to effect of hypoxia, MCF10A-ERBB2 cells expressing stable form of HIF-1α were able to maintain ERBB2, ERK, and AKT activation even when treated with high doses of lapatinib under normal oxygen conditions (Figure 4D). Stable HIF-1α expressing cells also contained reduced levels of BIM following lapatinib treatment compared to control cells (Figure 4D). Thus, HIF-1α expression alone is sufficient to block lapatinib-mediated effect on growth and signaling in ERBB2-expressing cells under normal oxygen conditions.


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

Karakashev SV, Reginato MJ - Oncotarget (2015)

HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B 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 4: HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).
Mentions: To determine whether HIF-1α stabilization is sufficient to confer lapatinib resistance in ERBB2-expressing cells under normoxic conditions, we overexpressed a stable non-degradable form of HIF-1α containing proline to alanine mutations (HIF-1α P402A, P564A) in MCF10A-ERBB2 cells. We confirmed that this mutant expressed levels similar to endogenous HIF-1α stabilized under hypoxia and to cells treated with prolyl-hydroxylase inhibitor DMOG (Figure 4A). MCF10A-ERBB2 cells expressing the stable form of HIF-1α were resistant to lapatinib-mediated effects on cell viability (Figure 4B) and cell number (Figure 4C) under normal oxygen conditions. In addition, similar to effect of hypoxia, MCF10A-ERBB2 cells expressing stable form of HIF-1α were able to maintain ERBB2, ERK, and AKT activation even when treated with high doses of lapatinib under normal oxygen conditions (Figure 4D). Stable HIF-1α expressing cells also contained reduced levels of BIM following lapatinib treatment compared to control cells (Figure 4D). Thus, HIF-1α expression alone is sufficient to block lapatinib-mediated effect on growth and signaling in ERBB2-expressing cells under normal oxygen conditions.

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