Investigating matriptase-directed 5mM NAC treatment resulted in increased apoptosis alone or in combination with ICI and/or Lapatinib in ER-positive breast cancer models.

100,000 A) BT474, B) MDA-MB-361 and C) MCF7 cells /well were seeded in 6-well plates and allowed to attach overnight. Cells were then treated with 5mM NAC, 5.4 uM Lapatinib, and 1uM ICI alone or in combination in serum-replete media for four days. NAC was administered again on day 3. Cells were collected and apoptosis determined by Annexin V/Propidium Iodide staining. Graphs are representative of 3 biological replicates. BT474 control (**, p = .0017 vs NAC), Lap (**, p = .0013 vs Lap+NAC), Lap+ICI (**, p = .0044 vs triple), MDA-MB-361 ICI (**, p = .0014 vs ICI+NAC), Lap (**, p = .0028 vs Lap+NAC), Lap+ICI (*, p = .0311 vs triple) MCF7 control (**, p = .0055 vs NAC), ICI (**, p = .0096 vs ICI/NAC), Lap/ICI (***, p = .0001 vs triple). Data shown as mean +/- SD of triplicate experiments;* p<0.05, ** p<0.01, ***p<.0.001.</p

Comparing methods of cellular stress showed that matriptase activation was not a hallmark of cellular stress but was robustly induced by etoposide.

100,000 BT474 cells/well were seeded into wells of a 6-well plate and allowed to attach overnight. Cells were then treated with 5.4uM Lapatinib and 1uM ICI in combination, 10uM Etoposide, 1uM Paclitaxel, 5.4uM Lapatinib alone, or 0.01% DMSO in either serum-replete or serum-free parallel experiments. Panels are representative of three biological replicates. Cells treated in serum-replete conditions were collected and A) assayed for apoptosis by Annexin V/Propidium Iodide staining and the conditioned media from cells treated in serum-free conditions was collected in order to B) determine levels of total and activated matriptase by immunoblotting with the total matriptase (M24) monoclonal antibody. Panels were the highest quality representative of 4 biological replicates.</p

Investigating matriptase-directed 5mM NAC treatment demonstrated increased apoptosis alone or in combination in HER2-amplified breast cancer cells.

100,000 A) AU565 and B) SKBr3 cells were seeded in 6-well plates and allowed to attach overnight. Cells were then treated with 5mM NAC, 5.4 uM Lapatinib and the combination of 10ug/ml Trastuzumab and 10ug/ml Pertuzumab alone or in combination in serum-replete media for four days. NAC was administered again on day 3. Cells were collected and apoptosis determined by Annexin V/Propidium Iodide staining. Graphs are representative of 3 biological replicates. AU565 control (***, pR Control (***, p = .0003 vs NAC), MTOR (*, p = .0125 vs combo). Data shown as mean +/- SD of triplicate experiments;* p<0.05, ** p<0.01, ***p<.0.001.</p

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Investigating endogenous, cell stress, and acid-induced matriptase activation demonstrated those mechanisms were mediated ROS and could be inhibited by N-acetyl cysteine (NAC).

A) SKBR3 cells were seeded in 35mm dishes and when ~80% confluent, washed twice with 1X sterile PBS and incubated for 3 hours with 5.4uM Lapatinib. Cells were then spiked with 10nm hydrogen peroxide for increasing amounts of time. Conditioned media was collected from the cells and analysed for levels of total and activated matriptase by the total matriptase (M24) antibody. B) MCF7 and C) BT474 cells were seeded in 35mm dishes and when 80% confluent. 5mM NAC was added to serum-replete media for 3 hours to pre-treat the cells. After pre-treatment, acid-induction buffer and 5mM NAC were added to cells either alone and in combination for 20 minutes. Cell lysates and conditioned media were collected in parallel and assayed for GAPDH as well as total and activated matriptase by the GAPDH and total matriptase (M24) antibody. D) MDA-MD-468, AU565, and SKBR3 cells were seeded in 35mm dishes and when ~80% confluent, incubated in 1ml serum-replete media with and without 5mM NAC for 24 hours. Conditioned media was collected from the cells and analysed for levels of total and activated matriptase by the total matriptase (M24) and activated matriptase (M69) antibody. Panels were representative of 3 biological replicates. E) AU565 cells were seeded in 35mm dishes and ~80% confluent, incubated in 1ml serum-replete media. Conditioned media was collected and 5mM NAC was added to experimental samples for 24 hours. The levels of activated matriptase were then assessed using total and activated matriptase antibodies. Panels were the highest quality representative of 4 biological replicates.</p

Investigating ICI and Lapatinib induced matriptase determined that only modest activation resulted from PI3K inhibition with no activation from MAPK inhibition and occurs as early as six hours post treatment.

A) BT474 and ZR-75-30 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then treated with 5.4uM Lapatinib, 10uM Trametinib, 10uM Pictilisib, or 0.01% DMSO. Conditioned media was collected with the appropriate lysis buffer (see methods). B) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then spiked with 5.4uM Lapatinib for increasing amounts of time. Cell lysates and conditioned media were collected with the appropriate lysis buffer. The levels of phospho-AKT, total and activated matriptase were determined by immunoblotting with p-S473 AKT and total matriptase (M24) monoclonal antibodies. C) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for increasing amounts of time in serum-free media with 1uM ICI. After each time point, cells were spiked with 5.4uM Lapatinib for 1 hour. Cell lysates were collected with the appropriate lysis buffer and the levels of total and activated matriptase were determined by immunoblotting with the total matriptase (M24) monoclonal antibody. GAPDH was used as a loading control. Panels were the highest quality representative of 4 biological replicates.</p

Matriptase expression and activation states.

Matriptase expression and activation states.</p

Screening breast cancer cells revealed robust matriptase activation only when ER-positive cells are treated with ICI and Lapatinib.

A) Breast cancer cells were seeded in 35mm dishes and when ~80% confluent, incubated for 24 hours in 1ml of basal media + 10% FBS. Conditioned media was collected and the level of activated matriptase was determined by immunoblotting with the total matriptase antibody. B) BT474 cells were seeded in 35mm dishes and when ~80% confluent, washed twice with sterile PBS, and pre-treated with 1uM ICI in serum-free media for 48 hours. Cells were then treated with either 20ng/ml EGF, 10nm estradiol (E2), 1uM ICI, or 5.4uM Lapatinib (Lap) with appropriate solvent controls for 48 hours. Conditioned media was collected and the level of activated matriptase was determined by immunoblotting with the activated matriptase antibody. Panels were the highest quality representative of 4 biological replicates.</p