ROS is a master regulator of in vitro matriptase activation.

Matriptase is a type II transmembrane serine protease that is widely expressed in normal epithelial cells and epithelial cancers. Studies have shown that regulation of matriptase expression and activation becomes deranged in several cancers and is associated with poor disease-free survival. Although the central mechanism of its activation has remained unknown, our lab has previously demonstrated that inflammatory conditions such as intracellular pH decrease strongly induces matriptase activation. In this investigation, we first demonstrate clear matriptase activation following Fulvestrant (ICI) and Tykerb (Lapatinib) treatment in HER2-amplified, estrogen receptor (ER)-positive BT474, MDA-MB-361 and ZR-75-30 or single ER-positive MCF7 cells, respectively. This activation modestly involved Phosphoinositide 3-kinase (PI3K) activation and occurred as quickly as six hours post treatment. We also demonstrate that matriptase activation is not a universal hallmark of stress, with Etoposide treated cells showing a larger degree of matriptase activation than Lapatinib and ICI-treated cells. While etoposide toxicity has been shown to be mediated through reactive oxygen species (ROS) and MAPK/ERK kinase (MEK) activity, MEK activity showed no correlation with matriptase activation. Novelly, we demonstrate that endogenous and exogenous matriptase activation are ROS-mediated in vitro and inhibited by N-acetylcysteine (NAC). Lastly, we demonstrate matriptase-directed NAC treatment results in apoptosis of several breast cancer cell lines either alone or in combination with clinically used therapeutics. These data demonstrate the contribution of ROS-mediated survival, its independence of kinase-mediated survival, and the plausibility of using matriptase activation to indicate the potential success of antioxidant therapy

Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation

We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Iβ2) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571–5577, 2000). Despite these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis

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

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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

Metabolic Maturation Exaggerates Abnormal Calcium Handling in a <i>Lamp2</i> Knockout Human Pluripotent Stem Cell-Derived Cardiomyocyte Model of Danon Disease

Danon disease (DD) is caused by mutations of the gene encoding lysosomal-associated membrane protein type 2 (LAMP2), which lead to impaired autophagy, glycogen accumulation, and cardiac hypertrophy. However, it is not well understood why a large portion of DD patients develop arrhythmia and sudden cardiac death. In the current study, we generated LAMP2 knockout (KO) human iPSC-derived cardiomyocytes (CM), which mimic the LAMP2 dysfunction in DD heart. Morphologic analysis demonstrated the sarcomere disarrangement in LAMP2 KO CMs. In functional studies, LAMP2 KO CMs showed near-normal calcium handling at base level. However, treatment of pro-maturation medium (MM) exaggerated the disease phenotype in the KO cells as they exhibited impaired calcium recycling and increased irregular beating events, which recapitulates the pro-arrhythmia phenotypes of DD patients. Further mechanistic study confirmed that MM treatment significantly enhanced the autophagic stress in the LAMP2 KO CMs, which was accompanied by an increase of both cellular and mitochondrial reactive oxygen species (ROS) levels. Excess ROS accumulation in LAMP2 KO CMs resulted in the over-activation of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and arrhythmogenesis, which was partially rescued by the treatment of ROS scavenger. In summary, our study has revealed ROS induced CaMKIIδ overactivation as a key mechanism that promotes cardiac arrhythmia in DD patients

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

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

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