Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 10;6(19):16902-11.
doi: 10.18632/oncotarget.4642.

ErbB-3 activation by NRG-1β sustains growth and promotes vemurafenib resistance in BRAF-V600E colon cancer stem cells (CSCs)

Affiliations

ErbB-3 activation by NRG-1β sustains growth and promotes vemurafenib resistance in BRAF-V600E colon cancer stem cells (CSCs)

Pramudita R Prasetyanti et al. Oncotarget. .

Abstract

Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1β-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1β strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1β-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.

Keywords: ErbB-3; NRG-1β; colon cancer stem cells; vemurafenib.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTERESTS

GS is an employee and shareholders of Mediapharma s.r.l.. SI is president and founder of Mediapharma s.r.l.. The other authors have no potential conflict of interest to disclose.

Figures

Figure 1
Figure 1. Effect of growth factors on wild-type and V600E-BRAF mutated colon CSCs
A. Cell proliferation was determined in wild-type (left panel) and BRAF mutant cells (right panel) in the presence or absence of growth factors (EGF and bFGF) by Cell Titer-Blue assay at the indicated time points. B. Basal or EGF-induced clonogenicity was assessed by limiting dilution assay in all cell lines. Proliferation evaluated by Cell Titer-Blue assay after 5 days of culture C. and clonogenicity evaluated by limiting dilution assay D. in BRAF mutant (Co123 and CC09) cells treated with Vemurafenib (1 μM) in the presence or absence of EGF. Data shown represent mean +/− SD from triplicate samples.
Figure 2
Figure 2. NRG-1β sustains proliferation of colon CSCs and reverts Vemurafenib antitumor effects
A. Surface expression level of ErbB receptors analyzed by FACS in both wt and BRAF mutant cells. B. Cell proliferation analysis of colon CSCs stimulated with increasing doses of NRG-1β evaluated by Cell Titer-Blue assay after 5 days of culture. C. CC09 cells were cultured in presence or absence of growth factors (EGF: 20 ng/ml; bFGF: 10 ng/ml; NRG-1β: 10 ng/ml) as indicated and proliferation assessed by cell count. D. Cell proliferation analysis evaluated by Cell Titer-Blue assay in BRAF mutant cells treated for 5 days with Vemurafenib in the presence of increasing amount of NRG-1β. E. Clonogenicity was evaluated by limiting dilution assay on BRAF mutant cells treated with Vemurafenib (1 μM) in presence or absence of NRG-1β.
Figure 3
Figure 3. ErbB-3 is required for NRG-1β-dependent escape to Vemurafenib
A. Immunoblot analysis of total and phosphorylated of ErbB-3 receptor and downstream signaling pathways evaluated in CC09 cells silenced for ErbB-3 (shErbB-3) as compared to same cells stably infected with the control vector (sh4Mut). After 24 hrs of growth factors deprivation, cells were stimulated with 10 ng/ml of NRG-1β for 5 minutes, then cell lysates were blotted as indicated. NRG-1β (10 ng/ml) stimulated clonogenicity was determined by limiting dilution assay in ErbB-3 silenced CC09 cells B. or in CC09 cells treated with 10 μg/ml of the anti-ErbB-3 antibody EV20 C. D. Co123 cells were cultured overnight in absence of growth factors and then treated with increasing doses of EV20 for 8 hrs before stimulation with NRG-1β (10 ng/ml) for 5 minutes; cell lysates were blotted as indicated. E. The rescue effect of NRG-1β (10 ng/ml) from Vemurafenib treatment (1 μM) was evaluated in CC09 and Co123 cells silenced for ErbB-3 (shErbB-3) as compared to control cells (sh4Mut). Proliferation was assessed by Cell Titer-Blue assay after 6 days of treatment either with NRG-1β (10 ng/ml) or Vemurafenib (1 μM) or a combination of the two. F. Proliferation, evaluated as in E, of BRAF mutant Co123 cells treated with Vemurafenib (1 μM) in the presence or absence of NRG-1β and EV20 (10 μg/ml). Results are expressed as mean +/− SD of two B. or three (C, E and F) independent experiments.* = p < 0.05, ** = p <0.01 (t-test).
Figure 4
Figure 4. Treatment with anti-ErbB-3 antibody results in delay of V600E-BRAF tumor growth
Tumor growth was assessed as described in Materials and Methods. A. Mice injected with CC09 (2.5 × 105) cells were divided in two groups one week after the engraftment. The treated group received 10 mg/kg twice weekly of EV20 in PBS whereas the control group received PBS only. Arrow indicates the start of treatment. B. Mice injected with either CC09 (2.5 × 105) or Co123 (1×106) cells and divided into size homogeneous groups once established tumors had reached the approximate Volume of 100 mm3, then treated with 10 mg/kg twice weekly of EV20. Control groups were treated with PBS. Results are expressed as Relative Tumor Volume, p values were determined by Student's t test and considered significant for p < 0.05.

Similar articles

Cited by

References

    1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. - PubMed
    1. Roskoski R. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res. 2014;79:34–74. - PubMed
    1. Schlessinger J. Receptor tyrosine kinases: legacy of the first two decades. Cold Spring Harb Perspect Biol. 2014:6. - PMC - PubMed
    1. Chong CR, Jänne PA. The quest to overcome resistance to EGFR-targeted therapies in cancer. Nat Med. 2013;19:1389–1400. - PMC - PubMed
    1. Tol J, Nagtegaal ID, Punt CJ. BRAF mutation in metastatic colorectal cancer. N Engl J Med. 2009;361:98–99. - PubMed

Publication types

MeSH terms