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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) regulates Bim in part through p38 mitogen-activated protein kinase (MAPK) activation. a UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were lysed. Lysates were probed with indicated antibodies. b UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were cultured in the presence of dimethyl sulfoxide (DMSO) or SB203580 (p38 inhibitor). Cells were lysed for 72 h (UACC893R1) or 96 h (MDA-MB-453) after infection, and lysates were probed with the indicated antibodies. Immunoprecipitation and western blot analysis were performed to show PTK6 downregulation in UACC893R1 cells; *PTK6 that was immunoprecipitated. c Cells expressing either control or PTK6 shRNAs (49, C9 or 12) were treated with either DMSO or SB203580. Cells were lysed 72 h (UACC893R1) or 120 h (MDA-MB-453) after shRNA lentiviral infection. Lysates were probed with the indicated antibodies. Experiments were performed three times
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Fig5: Protein tyrosine kinase 6 (PTK6) regulates Bim in part through p38 mitogen-activated protein kinase (MAPK) activation. a UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were lysed. Lysates were probed with indicated antibodies. b UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were cultured in the presence of dimethyl sulfoxide (DMSO) or SB203580 (p38 inhibitor). Cells were lysed for 72 h (UACC893R1) or 96 h (MDA-MB-453) after infection, and lysates were probed with the indicated antibodies. Immunoprecipitation and western blot analysis were performed to show PTK6 downregulation in UACC893R1 cells; *PTK6 that was immunoprecipitated. c Cells expressing either control or PTK6 shRNAs (49, C9 or 12) were treated with either DMSO or SB203580. Cells were lysed 72 h (UACC893R1) or 120 h (MDA-MB-453) after shRNA lentiviral infection. Lysates were probed with the indicated antibodies. Experiments were performed three times

Mentions: To determine the signaling pathways responsible for PTK6 downregulation-mediated Bim induction in UACC893R1 and MDA-MB-453 cells, we examined the status of major signaling pathways activated downstream of Her2 that have been implicated in survival, and Bim regulation [26, 28]. Interestingly, PTK6 downregulation did not consistently affect either Akt or Erk/mitogen-activated protein kinase (MAPK) signaling, two pathways known to regulate Bim expression (Fig. 5a). In contrast, we observed robust activation of p38 MAPK with PTK6 downregulation (Fig. 5a).Fig. 5


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) regulates Bim in part through p38 mitogen-activated protein kinase (MAPK) activation. a UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were lysed. Lysates were probed with indicated antibodies. b UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were cultured in the presence of dimethyl sulfoxide (DMSO) or SB203580 (p38 inhibitor). Cells were lysed for 72 h (UACC893R1) or 96 h (MDA-MB-453) after infection, and lysates were probed with the indicated antibodies. Immunoprecipitation and western blot analysis were performed to show PTK6 downregulation in UACC893R1 cells; *PTK6 that was immunoprecipitated. c Cells expressing either control or PTK6 shRNAs (49, C9 or 12) were treated with either DMSO or SB203580. Cells were lysed 72 h (UACC893R1) or 120 h (MDA-MB-453) after shRNA lentiviral infection. Lysates were probed with the indicated antibodies. Experiments were performed three times
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496943&req=5

Fig5: Protein tyrosine kinase 6 (PTK6) regulates Bim in part through p38 mitogen-activated protein kinase (MAPK) activation. a UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were lysed. Lysates were probed with indicated antibodies. b UACC893R1 or MDA-MB-453 cells expressing either control or PTK6 shRNA (49, C9 or 12) were cultured in the presence of dimethyl sulfoxide (DMSO) or SB203580 (p38 inhibitor). Cells were lysed for 72 h (UACC893R1) or 96 h (MDA-MB-453) after infection, and lysates were probed with the indicated antibodies. Immunoprecipitation and western blot analysis were performed to show PTK6 downregulation in UACC893R1 cells; *PTK6 that was immunoprecipitated. c Cells expressing either control or PTK6 shRNAs (49, C9 or 12) were treated with either DMSO or SB203580. Cells were lysed 72 h (UACC893R1) or 120 h (MDA-MB-453) after shRNA lentiviral infection. Lysates were probed with the indicated antibodies. Experiments were performed three times
Mentions: To determine the signaling pathways responsible for PTK6 downregulation-mediated Bim induction in UACC893R1 and MDA-MB-453 cells, we examined the status of major signaling pathways activated downstream of Her2 that have been implicated in survival, and Bim regulation [26, 28]. Interestingly, PTK6 downregulation did not consistently affect either Akt or Erk/mitogen-activated protein kinase (MAPK) signaling, two pathways known to regulate Bim expression (Fig. 5a). In contrast, we observed robust activation of p38 MAPK with PTK6 downregulation (Fig. 5a).Fig. 5

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