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. 2025 Jan;45(1):68-73.
doi: 10.1002/cac2.12632. Epub 2024 Nov 24.

PTPN9 regulates HER3 phosphorylation during trastuzumab treatment and loss of PTPN9 is a potential biomarker for trastuzumab resistance in HER2 positive breast cancer

Abul Azad  1   2   3 , Maryam Arshad  2   3 , Daniele Generali  4 , Katharina Feldinger  1 , Merel Gijsen  1 , Carla Strina  4 , Mariarosa Cappelletti  4 , Daniele Andreis  4   5 , Russell Leek  1 , Syed Haider  1   6 , Pirkko-Liisa Kellokumpu-Lehtinen  7 , Ioannis Roxanis  6   8 , Adrian Llewellyn Harris  1 , Abeer Mahmoud Shaaban  9 , Heikki Joensuu  10 , Anthony Kong  1   2   3
Affiliations

PTPN9 regulates HER3 phosphorylation during trastuzumab treatment and loss of PTPN9 is a potential biomarker for trastuzumab resistance in HER2 positive breast cancer

Abul Azad et al. Cancer Commun (Lond). 2025 Jan.
No abstract available

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Conflict of interest statement

The authors declare no competing financial interests but the following competing non‐financial interests: Dr. Anthony Kong filed a patent of PTPN9 as a biomarker in relation to cancer treatment (international patent application No. PCT/GB2013/050057). However, this patent was not subsequently being maintained by further payments. All other authors declare no competing financial or non‐financial interests.

Figures

FIGURE 1
FIGURE 1
Trastuzumab upregulates PTPN9 which is associated with HER3 dephosphorylation and PTPN9 is a potential prognostic biomarker in HER2 positive breast cancer patients. (A) Both SKBR3 and BT474 cells were treated with 40 µg/mL of trastuzumab for indicated times and were assayed for expression and phosphorylation of indicated proteins by western blot. Immunoblots were also performed for indicated proteins from both trastuzumab‐resistant SKBR3 and BT474 cells and after withdrawal of trastuzumab from resistant cells for 24 hours. Actin represents equal loading control. (B) SKBR3 and BT474 cells were transfected with 50 nmol/L of control siRNA (csiRNA) and two independent specific siRNA against PTPN9. After a total of 48 hours’ transfection, cells were treated with trastuzumab for 4 and 24 hours. (C) SKBR3 cells were transfected with wild type (WT) and mutant PTPN9 overexpressing plasmids. Following transfection and selection with G418, the cells were seeded and treated with trastuzumab for 72 hours. The cells were lysed and analyzed for expression of PTPN9 and level of HER3 and HER2 phosphorylation. (D) Similarly, trastuzumab‐resistant SKBR3 cells were also transfected with WT and mutant PTPN9 overexpressing plasmids and were analyzed for expression of indicated proteins. In all cases, actin was used as loading control. (E) Interaction studies between PTPN9 and HER family receptors were performed in SKBR3 and BT474 cells ± trastuzumab for 4 hours and also in trastuzumab‐resistant SKBR3 and BT474 cells. Immunoprecipitations was performed with anti‐HER3, anti‐HER2, or anti‐EGFR antibodies before blotted for PTPN9 (top panels). In each case, immunoprecipitation was also blotted for the respective antibody to show equal immunoprecipitation was performed. IgG was used as a IP control. 5% of total extracts were used as input. (F) Immunoprecipitations was performed with anti‐PTPN9 antibody using cell lysates from SKBR3 cells transfected with empty vector (EV), WT PTPN9, mutant C515S and D470A forms of PTPN9 and were blotted for anti‐HER3 or anti‐pHER3 antibody. Immunoprecipitation was also done with the same antibody to show that equal immunoprecipitation was performed. IgG used as an IP control. 10% of total extracts was used as input. (G) Patient‐derived organoids (PDOs) from HER2 ve+ (left), normal tissue (right) were treated 40 ug/mL of trastuzumab for indicated times. Expression of indicated proteins were analysed by immunoblot. (H) Schematic illustrations of the regulation of HER3 signalling by PTPN9 in relation to trastuzumab treatment in HER2 positive breast cancer cells: in the parental SKBR3/BT474 cells, HER receptors dimerize with each other upon growth factor stimulation, resulting in the activation of HER3 and its downstream activator Akt, which leads to proliferation and survival. Following trastuzumab treatment in the parental SKBR3 cells, PTPN9 expression is upregulated. It interacts with EGFR, HER2 and HER3 although it only dephosphorylates HER3, leading to a decrease in Akt phosphorylation. In chronic exposure to trastuzumab leading to its acquired resistance, PTPN9 expression is decreased again, resulting in the reactivation of HER3 and its downstream effector Akt. Following an overexpression of PTPN9 in trastuzumab‐resistant cells, HER3 could be dephosphorylated again leading to a decrease in Akt phosphorylation. (I) Using the established IRS scoring system, the breast cancer patients from a cohort in Oxford (n = 59) were split into two groups according to PTPN9 IRS scoring of either 4 (n = 50) and higher or below 4 (n = 9). PTPN9 expression was split into low and high expression and the relapse‐free survival (Left) and overall survival (Right) between the two groups was assessed by Log‐rank test. (J) Similarly, PTPN9 expression in relation to overall survival between the two groups was also assessed in all (Left upper panel), trastuzumab treated (Right upper panel) and non‐trastuzumab treated (Left lower panel) of HER2 positive patients in FinHER trial. PTPN9 expression in relation to distant recurrence‐free survival between the two groups was further assessed in HER2‐positive FinHER patients. (Right lower panel) PTPN9 expression was split into low and high expression and the time‐to‐distant recurrences between the two groups for all FinHER patients was assessed by Log‐rank test. Abbreviations: T‐DM1, ado‐trastuzumab emtansine; T‐Dxd, trastuzumab deruxtecan; PDOs, patient‐derived organoids; IRS, immunoreactive scoring system; TMA, tissue microarray, PTPN9, tyrosine‐protein phosphatase non‐receptor type 9; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; ADAM10/17, a disintegrin and metalloproteinase 10/17.
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