The role of stem cells in glioma progression and therapy

The concepts of tumour origin and stochastic nature of carcinogenesis are being challenged today by hierarchical models that predict the existence of cancer stem cells (CSCs), which are postulated as unique cell population capable of infinite self renewal, multilineage differentiation and having a higher resistance to conventional cancer therapy thus facilitating malignant growth and therapy resistance. Accordingly, successful treatment of adult brain tumourglioma and its most malignant stageglioblastoma multiforme (GBM), would require the elimination of CSCs to avoid tumour relapse. Yet, with available therapy (i.e. surgery) in GBMs this cannot be achieved, due to infiltrative growth of a subpopluation of GBM cells with highly expressed migratory genes (migratome) into the normal brain tissue. Besides CSCs a proven prerequisite for tumour development and progression, tumour bulk mass also comprises haematopoietic stem cells, endothelial progenitor cells and mesenchymal stem cells (MSCs). The role of these other types of stem cell was shown to largely depend on the tumour microenvironment, where their contradictory anti-tumour action was evidenced. Yet, the exact mechanisms and MSCs role in cell-mediated modulation of tumour behaviour via paracrine and direct interactions with GBM (stem) cells still remain unknown. Nevertheless these stem cells, particularly MSCs, may represent novel therapeutic vectors for enhanced target-site delivery of chemotherapeutics, which are urgently needed to improve efficiency of current glioma treatment. So far, cell therapy using MSCs appears promising, due to MSCs selective tumour tropism and their immuno-modulatory potential regarding treatment of GBM, which will be discussed in this review

Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression

Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts, appeared to be overexpressed as well in various types of cancers. In this review, the physiological functions of CatK are presented and compared to its potential role in pathobiolology of processes associated with tumour growth, invasion and metastasis of cancer cells and their interactions with the tumour microenvironment. CatK activity is either indirectly affecting signalling pathways, or directly degrading extracellular matrix (ECM) proteins, for example in bone metastases. Recently, CatK was also found in glioma, possibly regulating cancer stem-like cell mobilisation and modulating recently found physiological CatK substrates, including chemokines and growth factors. Moreover, CatK may be useful in differential diagnosis and may have prognostic value. Finally, the application of CatK inhibitors, which are already in clinical trials for treatment of osteoporosis, has a potential to attenuate cancer aggressivenes

La Vigie marocaine

08 avril 19361936/04/08 (A27,N8879)-1936/04/08

Scheme of cellular processes and activities involving overexpressed protease and protease inhibitor genes in GBM.

<p>The overexpressed protease and inhibitor genes in GBM tissues and cells were queried by the Biomine search engine which identified processes and activities ascribed with KEGG and GO identifiers (in circles) in which selected genes (in bold caption) are involved.</p

Signaling pathways in which the candidate genes are involved.

<p>An extensive literature search revealed that the candidate genes are cross-linked to 3 signaling pathways: NF-κB, Akt and MAPK, which all play a role in cancer. NF-κB signaling pathway has a crucial role in regulating immune responses, whereas Akt signaling has been shown to inhibit the growth of GBM cells and GBM stem-like cells that may also be impaired by MAPK signaling disruption. Because of the RT-qPCR results, <i>CTSK</i>'s role has been examined and it was found via cross linking to other candidate genes obtained via osteopontin (<i>OPN</i>) gene functions.</p

RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain <i>(in vivo)</i>.

<p>(A) Upregulated expression of seven genes (<i>TFRC</i>, <i>CTSK</i>, <i>GFPT2</i>, <i>ERAP2</i>, <i>GPR56</i>, <i>CD74</i>, <i>PI3</i>) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the <i>CTSK</i> gene using GBM tissues and cell lines with reference genes <i>TBP</i> and <i>HPRT1</i> in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.</p

Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation

Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.

<p>ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. <b>Please note</b> that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.</p