Ann-Charlotte Johansson

Regulation of apoptosis-associated lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine

AbstractThere is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 μM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

Lysosomal membrane permeabilization during apoptosis--involvement of Bax?

Bcl‐2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis‐inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co‐localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno‐electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Cytosolic acidification and lysosomal alkalinization during TNF-α induced apoptosis in U937 cells

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-α, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 ± 0.1 in healthy cells to 6.8 ± 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 ± 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F0F1-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 ± 0.3, in the healthy population, to 4.8 ± 0.3 and 5.5 ± 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-α.

Cancer-Associated Fibroblasts Induce Matrix Metalloproteinase–Mediated Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma Cells

A growing body of evidence suggests that components of the tumor microenvironment, including cancer-associated fibroblasts (CAF), may modulate the treatment sensitivity of tumor cells. Here, we investigated the possible influence of CAFs on the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to cetuximab, an antagonistic epidermal growth factor receptor (EGFR) antibody. Cetuximab treatment caused a reduction in the proliferation rate of HNSCC cell lines, whereas the growth of HNSCC-derived CAF cultures was unaffected. When tumor cells were cocultured with CAFs in a transwell system, the cetuximab-induced growth inhibition was reduced, and a complete protection from growth inhibition was observed in one of the tumor cell lines investigated. Media that had been conditioned by CAFs offered protection from cetuximab treatment in a concentration-dependent manner, suggesting that the resistance to treatment was mediated by CAF-derived soluble factors. The coculture of HNSCC cell lines with CAFs resulted in an elevated expression of matrix metalloproteinase-1 (MMP-1) in both the tumor cells and CAFs. Moreover, the CAF-induced resistance was partly abolished by the presence of an MMP inhibitor. However, CAFs treated with siRNA targeting MMP-1 still protected tumor cells from cetuximab treatment, suggesting that several MMPs may cooperate to facilitate resistance or that the protective effect is mediated by another member of the MMP family. These results identify a novel CAF-dependent modulation of cetuximab sensitivity and suggest that inhibiting MMPs may improve the effects of EGFR-targeted therapy. Mol Cancer Res; 10(9); 1158–68. ©2012 AACR.

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44high/EGFRlow, CD44high/EGFRhigh and CD44low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44high/EGFRlow population showed a spindle-shaped EMT-like morphology, while the CD44low population was dominated by cobblestone-shaped cells. The CD44high/EGFRlow population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44high/EGFRlow cells in comparison to CD44low cells. Moreover, CD44high/EGFRlow cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44high/EGFRhigh and CD44low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.

EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines

BACKGROUND Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. METHODS Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. RESULTS Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). CONCLUSIONS Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

YAP1 is a potential biomarker for cetuximab resistance in head and neck cancer

OBJECTIVES Targeted therapy against the epidermal growth factor receptor (EGFR) only variably represents a therapeutic advance in head and neck squamous cell carcinoma (HNSCC). This study addresses the need of biomarkers of treatment response to the EGFR-targeting antibody cetuximab (Erbitux®). MATERIALS AND METHODS The intrinsic cetuximab sensitivity of HNSCC cell lines was assessed by a crystal violet assay. Gene copy number analysis of five resistant and five sensitive cell lines was performed using the Affymetrix SNP 6.0 platform. Quantitative real-time PCR was used for verification of selected copy number alterations and assessment of mRNA expression. The functional importance of the findings on the gene and mRNA level was investigated employing siRNA technology. The data was statistically evaluated using Mann-Whitney U-test and Spearmans correlation test. RESULTS Analysis of the intrinsic cetuximab sensitivity of 32 HNSCC cell lines characterized five and nine lines as cetuximab sensitive or resistant, respectively. Gene copy number analysis of five resistant versus five sensitive cell lines identified 39 amplified protein-coding genes, including YAP1, in the genomic regions 11q22.1 or 5p13-15. Assessment using qPCR verified that YAP1 amplification associated with cetuximab resistance. Amplification of YAP1 correlated to higher mRNA levels, and RNA knockdown resulted in increased cetuximab sensitivity. Assessment of several independent clinical data sets in the public domain confirmed YAP1 amplifications in multiple tumor types including HNSCC, along with highly differential expression in a subset of HNSCC patients. CONCLUSION Taken together, we provide evidence that YAP1 could represent a novel biomarker gene of cetuximab resistance in HNSCC cell lines.

Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines.

BACKGROUND Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines. METHODS The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-X(L) , cyclooxygenase-2 (COX-2), and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse. RESULTS Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (IR; r = 0.36, P = 0.02). The combination of survivin, Bax, Bcl-2, Bcl-X(L) , COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r = 0.553, P < 0.001). CONCLUSION These data indicate that the IR of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.

Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells.

Conclusion. Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines. Objective. To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis. Material and methods. Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA. Results. Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.