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PTK6 inhibition promotes apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by inducing Bim.

Park SH, Ito K, Olcott W, Katsyv I, Halstead-Nussloch G, Irie HY - Breast Cancer Res. (2015)

Bottom Line: We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death.Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Medical Oncology, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 1468 Madison Avenue, New York, NY, USA. sun.park@mssm.edu.

ABSTRACT

Introduction: Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2(+) (Her2(+)) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2(+) breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2(+) breast cancer, either intrinsically or acquired after continuous drug exposure.

Methods: To determine the effects of PTK6 inhibition on Lapatinib-resistant Her2(+) breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.

Results: Lapatinib treatment of "sensitive" Her2(+) cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively "resistant" Her2(+) cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D Matrigel(TM) cultures, and also inhibits growth of Her2(+) primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.

Conclusions: PTK6 downregulation induces apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.

No MeSH data available.


Related in: MedlinePlus

Protein tyrosine kinase 6 (PTK6) downregulation inhibits growth of Lapatinib-resistant human epidermal growth factor receptor 2 (Her2)+ breast cancer cells. a UACC893R1 or MDA-MB-453 cells grown in monolayer cultures expressing control or PTK6 shRNA (C9), were counted at the indicated number of days. b Cells expressing either control or two different PTK6 shRNAs (C9, 49) were grown in 3-D MatrigelTM cultures. Cells were re-fed with fresh media every 3–4 days. The figure represents day-16 cultures (UACC893R1) or day-23 cultures (MDA-MB-453). Scale bar 30 μm. c Cells expressing control or PTK6 shRNA (C9, 49) were cultured in soft agar assays for 30 days. Colonies greater than 50 square pixels were counted and plotted. Downregulation of PTK6 was confirmed by western analyses for MDA-MB-453 and UACC893R1 cells. d UACC893R1 cells expressing control or PTK6 shRNA (C9) were injected subcutaneously into flanks of 6-week-old female nude mice (n = 5/group). Tumor size was measured and recorded every 3–4 days. All figures are representative of three independent experiments except for the xenograft study, which was performed twice; *p <0.05; **p <0.005
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Fig2: Protein tyrosine kinase 6 (PTK6) downregulation inhibits growth of Lapatinib-resistant human epidermal growth factor receptor 2 (Her2)+ breast cancer cells. a UACC893R1 or MDA-MB-453 cells grown in monolayer cultures expressing control or PTK6 shRNA (C9), were counted at the indicated number of days. b Cells expressing either control or two different PTK6 shRNAs (C9, 49) were grown in 3-D MatrigelTM cultures. Cells were re-fed with fresh media every 3–4 days. The figure represents day-16 cultures (UACC893R1) or day-23 cultures (MDA-MB-453). Scale bar 30 μm. c Cells expressing control or PTK6 shRNA (C9, 49) were cultured in soft agar assays for 30 days. Colonies greater than 50 square pixels were counted and plotted. Downregulation of PTK6 was confirmed by western analyses for MDA-MB-453 and UACC893R1 cells. d UACC893R1 cells expressing control or PTK6 shRNA (C9) were injected subcutaneously into flanks of 6-week-old female nude mice (n = 5/group). Tumor size was measured and recorded every 3–4 days. All figures are representative of three independent experiments except for the xenograft study, which was performed twice; *p <0.05; **p <0.005

Mentions: We determined the effects of downregulating PTK6 expression on the growth and survival of MDA-MB-453 or UACC893R1 cells. ShRNA-vector-mediated downregulation of PTK6 alone significantly inhibited growth in 2-D monolayer and 3-D Matrigel culturesTM (Fig. 2a, b). PTK6 shRNA expression impaired soft agar colony formation of MDA-MB-453 and UACC893R1 cells (Fig. 2c). PTK6 downregulation also suppressed growth of UACC893R1 primary tumor xenografts (Fig. 2d). The compromised growth of these Lapatinib-resistant Her2+ cells in 2-D and 3-D cultures and in vivo is in part due to increased apoptosis; PTK6 shRNA expression enhanced the percentage of cells in the sub-G1 fraction, and Annexin-V-positive cells (Fig. 3a, b and Additional file 3: Figure S3A). In addition, PTK6 downregulation enhanced levels of cleaved PARP (Fig. 3c and Additional file 3: Figure S3B). These data support apoptosis induction as a mechanism by which PTK6 downregulation impairs survival and growth of Lapatinib-resistant Her2+ breast cancer cells.Fig. 2


PTK6 inhibition promotes apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by inducing Bim.

Park SH, Ito K, Olcott W, Katsyv I, Halstead-Nussloch G, Irie HY - Breast Cancer Res. (2015)

Protein tyrosine kinase 6 (PTK6) downregulation inhibits growth of Lapatinib-resistant human epidermal growth factor receptor 2 (Her2)+ breast cancer cells. a UACC893R1 or MDA-MB-453 cells grown in monolayer cultures expressing control or PTK6 shRNA (C9), were counted at the indicated number of days. b Cells expressing either control or two different PTK6 shRNAs (C9, 49) were grown in 3-D MatrigelTM cultures. Cells were re-fed with fresh media every 3–4 days. The figure represents day-16 cultures (UACC893R1) or day-23 cultures (MDA-MB-453). Scale bar 30 μm. c Cells expressing control or PTK6 shRNA (C9, 49) were cultured in soft agar assays for 30 days. Colonies greater than 50 square pixels were counted and plotted. Downregulation of PTK6 was confirmed by western analyses for MDA-MB-453 and UACC893R1 cells. d UACC893R1 cells expressing control or PTK6 shRNA (C9) were injected subcutaneously into flanks of 6-week-old female nude mice (n = 5/group). Tumor size was measured and recorded every 3–4 days. All figures are representative of three independent experiments except for the xenograft study, which was performed twice; *p <0.05; **p <0.005
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Fig2: Protein tyrosine kinase 6 (PTK6) downregulation inhibits growth of Lapatinib-resistant human epidermal growth factor receptor 2 (Her2)+ breast cancer cells. a UACC893R1 or MDA-MB-453 cells grown in monolayer cultures expressing control or PTK6 shRNA (C9), were counted at the indicated number of days. b Cells expressing either control or two different PTK6 shRNAs (C9, 49) were grown in 3-D MatrigelTM cultures. Cells were re-fed with fresh media every 3–4 days. The figure represents day-16 cultures (UACC893R1) or day-23 cultures (MDA-MB-453). Scale bar 30 μm. c Cells expressing control or PTK6 shRNA (C9, 49) were cultured in soft agar assays for 30 days. Colonies greater than 50 square pixels were counted and plotted. Downregulation of PTK6 was confirmed by western analyses for MDA-MB-453 and UACC893R1 cells. d UACC893R1 cells expressing control or PTK6 shRNA (C9) were injected subcutaneously into flanks of 6-week-old female nude mice (n = 5/group). Tumor size was measured and recorded every 3–4 days. All figures are representative of three independent experiments except for the xenograft study, which was performed twice; *p <0.05; **p <0.005
Mentions: We determined the effects of downregulating PTK6 expression on the growth and survival of MDA-MB-453 or UACC893R1 cells. ShRNA-vector-mediated downregulation of PTK6 alone significantly inhibited growth in 2-D monolayer and 3-D Matrigel culturesTM (Fig. 2a, b). PTK6 shRNA expression impaired soft agar colony formation of MDA-MB-453 and UACC893R1 cells (Fig. 2c). PTK6 downregulation also suppressed growth of UACC893R1 primary tumor xenografts (Fig. 2d). The compromised growth of these Lapatinib-resistant Her2+ cells in 2-D and 3-D cultures and in vivo is in part due to increased apoptosis; PTK6 shRNA expression enhanced the percentage of cells in the sub-G1 fraction, and Annexin-V-positive cells (Fig. 3a, b and Additional file 3: Figure S3A). In addition, PTK6 downregulation enhanced levels of cleaved PARP (Fig. 3c and Additional file 3: Figure S3B). These data support apoptosis induction as a mechanism by which PTK6 downregulation impairs survival and growth of Lapatinib-resistant Her2+ breast cancer cells.Fig. 2

Bottom Line: We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death.Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Medical Oncology, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 1468 Madison Avenue, New York, NY, USA. sun.park@mssm.edu.

ABSTRACT

Introduction: Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2(+) (Her2(+)) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2(+) breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2(+) breast cancer, either intrinsically or acquired after continuous drug exposure.

Methods: To determine the effects of PTK6 inhibition on Lapatinib-resistant Her2(+) breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.

Results: Lapatinib treatment of "sensitive" Her2(+) cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively "resistant" Her2(+) cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these "resistant" cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D Matrigel(TM) cultures, and also inhibits growth of Her2(+) primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.

Conclusions: PTK6 downregulation induces apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.

No MeSH data available.


Related in: MedlinePlus