Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma

Abstract

Background: Kallikreins have prognostic value in specific malignancies, but few studies have addressed their clinical significance to glioblastoma multiforme (GBM). Kallikrein 6 (KLK6) is of potential high relevance to GBM, since it is upregulated at sites of CNS pathology and linked to reactive astrogliosis. Here we examine the clinical value of KLK6 as a prognostic indicator of GBM patient survival and its activity in promoting resistance to cytotoxic agents.

Methods: The association between patient survival and levels of KLK6 immunoreactivity were investigated in 60 grade IV astrocytoma tumor specimens. Levels of KLK6 RNA were also evaluated in a separate set of GBM patient tumors (n = 23). Recombinant KLK6 or enforced KLK6 overexpression in GBM cell lines was used to evaluate effects on astrocytoma cell survival.

Results: A range of KLK6 expression was observed across grade IV tumors, with higher levels a poor prognostic indicator of patient survival (P = .02) even after adjusting for gender and Eastern Cooperative Oncology Group performance scores (P = .01). KLK6 reduced the sensitivity of GBM cell lines to cytotoxic agents, including staurosporine and cisplatin, and to the current standard of patient care: radiotherapy or temozolomide alone or in combination. The ability of KLK6 to promote resistance to apoptosis was dependent on activation of the thrombin receptor, protease activated receptor 1.

Conclusions: Taken together, these results indicate that elevated levels of KLK6 in GBM are likely to promote the resistance of tumor cells to cytotoxic agents and are an indicator of reduced patient postsurgical survival times.

Fig. 1.

KLK6 is differentially expressed across grade III and grade IV astrocytoma and is associated with poor patient survival. Tissue microarrays containing grade III (A) and grade IV (B) surgically resected astrocytoma samples were evaluated for KLK6 IR (grade III 2.4 ± 1.5, grade IV 7.7 ± 2.4; mean ± SD), P < .001, 2-sample t-test (see Supplementary material, Tables S1–S3). Photomicrographs captured with the Bliss digital imaging system (20× objective, C–G) or using 100× oil (H–L) show examples of the range of KLK6 IR seen in grade III IR = 1.5 (C and H) or IR = 6 (D and I), and in grade IV IR = 2.5 (E and J), IR = 7 (F and K), or IR = 12 (G and L) astrocytoma specimens (scale bar, 100 µm). In the higher power images, the hematoxylin-stained nuclei can be readily distinguished from KLK6 IR seen in association with cellular cytoplasm or the surrounding matrix. (M) Kaplan–Meier curves for KLK6 IR scores of <10 or ≥10 in grade IV patients (P = .014, univariable analysis; see Table 1). (N) Histogram shows level of KLK6 RNA in nondiseased control human brain samples (mean 0.86 ± 0.3 SEM) and in grade IV tumor specimens (mean 21.1 ± 3.8 SEM), P < .05, 2-sample Student's t-test.

Fig. 2.

Recombinant KLK6 promotes the resistance of astrocytoma cell lines to staurosporine-induced cell death. (A) Treatment of U251 astrocytoma cells with 5 or 10 µg/mL of recombinant KLK6 significantly reduced the number of PI-labeled dead cells detected 18 h after the addition of staurosporine (1.0 µM). In the same experiment, 1.7% of untreated U251 cells were PI labeled. Data are mean ± SEM of triplicate cultures examined in parallel, *P = .02, 2-sample Student's t-test. Dot blots (B) show representative examples of flow cytometry data demonstrating the reduction in the percentage of PI-labeled dead cells seen in response to increasing concentrations of KLK6 (SSC, side scatter). (C) In parallel, treatment of SF767 astrocytoma cells with 10 µg/mL recombinant KLK6 significantly reduced the number of PI-labeled dead cells detected 18 h after addition of staurosporine (1.0 µM), **P = .001, 2-sample Student's t-test; 1.6% of untreated SF767 cells were PI labeled in the same experiment.

Fig. 3.

Recombinant KLK6 promotes resistance of U251 astrocytoma cells to apoptosis induced by staurosporine. Histogram (A) and corresponding dot plots (B) demonstrate that KLK6 significantly increases the number of live cells (annexin V–FITC⁻ and PI⁻), while reducing the number of early apoptotic (annexin V–FITC⁺ and PI⁻) and dead cells (annexin V–FITC⁺ and PI⁺) seen in response to 1 µM staurosporine. Data shown are mean ± SEM of triplicate cultures examined in parallel, *P = .04, **P ≤ .001, 2-sample Student's t-test. In the same experiment, 82.8% of untreated U251 cells were annexin V–FITC⁻ and PI⁻, 0.7% were annexin V–FITC⁺ and PI⁻, and 16% were annexin V–FITC⁺ and PI⁺. Western blot (C) shows increased levels of cleaved PARP (full-length 116 kDa, cleaved 89 kDa) after treatment with staurosporine (ST; 2 μM, 6 h). Pretreatment of cultures for 1 h with KLK6 (10 μg/mL) prior to application of ST reduced the amount of cleaved PARP observed (55% reduction; P = .01, Student's t-test). Actin was used as a control for loading, and results shown are representative of those from 3 separate experiments.

Fig. 4.

Knockdown of PAR1 blocks KLK6-mediated rescue of staurosporine-treated U251 astrocytoma cells. (A) U251 cells were stably transfected with a vector coding for an shRNA specific for human PAR1 (shPAR1-3 or shPAR1-4) or a no target control vector, and the ability of KLK6 (5 µg/mL) to reduce staurosporine (1 µM)-induced cell death was examined. Histogram shows that the ability of recombinant KLK6 to reduce cell death elicited by staurosporine was reduced by both PAR1-targeting vectors but only significantly blocked by the shPAR1-4 vector that produced the highest levels of knockdown (65%). In this experiment, untreated control cells contained 1.9%, 2.0%, and 1.9% PI⁺ cells in the case of no target control, shPAR1-3, and shPAR1-4 cells, respectively. (B) Knockdown of PAR1 RNA was determined by real-time RT-PCR (ratio of PAR1 copy number to GAPDH copy number calculated in each case and expressed as percent control). Data shown are mean ± SEM of triplicate samples examined in parallel, 2-sample Student's t-test: *P < .03, **P < .015.

Fig. 5.

Overexpression of KLK6 in U251 astrocytoma cells promotes resistance to staurosporine-induced cell death. (A and B) U251 astrocytoma cells were stably transfected with a vector in which the human KLK6 gene was constitutively expressed N-terminal to GFP under the control of a CMV promoter (KLK6-CMV) or with an empty vector (Control-CMV) and examined for cell death in response to staurosporine (2.0 µM). Relative to controls, astrocytoma cells engineered to overexpress KLK6 showed significant reductions in staurosporine-induced cell death measured by uptake of 7-AAD using flow cytometry (SSC, side scatter). In this experiment, untreated Control-CMV and KLK6-CMV U251 cultures contained 2.1% 7-AAD⁺ cells. (C) Histogram shows ratio of the level KLK6 RNA to that of the constitutively expressed gene GAPDH in control and stably transfected U251 cells (A to C, KLK6-CMV-Clone 1). (D–K) Photomicrographs show the localization of GFP in U251 astrocytoma cells transfected with control (D–G) or the KLK6-CMV expression construct (H–K, KLK6-CMV-Clone 3). U251 cells were co-labeled with rhodamine-conjugated phalloidin to visualize actin (E and I) and counterstained with DAPI (4’,6-diamidino-2-phenylindole; F and J) to enumerate nuclei. Merged images are also shown (G and K). (K, scale bar = 100 µm for all images.) Data shown are mean ± SEM of triplicate samples examined in parallel, 2-sample Student's t-test: *P = .02 (A), **P = .001 (C). Results are representative of those seen in at least 3 independent cell culture experiments.

Fig. 6.

Overexpression of KLK6 in U251 astrocytoma cells promotes cell survival and resistance to cell death induced by radiotherapy or TMZ alone or in combination. Clonogenic assays were performed to determine the effect of KLK6 overexpression (KLK6-CMV) on the survival of U251 astrocytoma cells and their sensitivity to 2 or 5 Gy of radiation, to 10 µM TMZ, or to 2 Gy of radiation + 10 µM TMZ. The number of colonies formed in each case was directly compared with nontransfected control U251 cells and with U251 cells stably expressing an empty control vector (Control-CMV). In each case, plated cells (1 × 10³ cells per well, A and B; 2 × 10³ cells per well, C and D; 5 × 10³ cell per well, E and F) were treated over a 24-h period and then allowed to expand for 2 wk prior to being fixed and stained with crystal violet. (A, C, and E) Histograms show the number of surviving colonies (± SEM) in triplicate; cultures treated in parallel expressed as a percent of cells plated initially. Mean colony counts ± SEM are listed for each cell line and treatment in Table 2. A significant increase in colony number was observed in KLK6-overexpressing cells under all treatment conditions examined (*P ≤ .02, **P ≤ .0005, 2-sample Student's t-test). (B, D, and F) Images of representative crystal violet stained colonies are shown for each treatment condition. Results shown were obtained using KLK6-CMV-Clone 1 (A and B), -Clone 2 (C and D), and -Clone 3 (E and F).

Fig. 7.

Overexpression of KLK6 promotes resistance of U251 astrocytoma cells to cell death induced by cisplatin. (A) Histogram shows the percent of cell death (7-AAD⁺ by flow cytometry) after exposure of U251 astrocytoma cells stably expressing an empty control CMV vector or a vector in which KLK6 is constitutively expressed under the control of CMV (KLK6-CMV-Clone 4) to cisplatin (2.5 μg/mL, 48 h). In the experiment shown in (A), untreated Control-CMV cultures contained 1.1% 7-AAD⁺ cells, while untreated KLK6-CMV cultures contained 1.9% 7-AAD⁺ cells. (B and C) U251 astrocytoma cells overexpressing KLK6 (KLK6-CMV-Clone 3) also showed significantly higher levels of survival when plated at a density of 2 × 10³ cells per well, transiently treated with cisplatin (0.1 μg/mL, 24 h), and then allowed to expand for 2 wk in culture before being fixed and stained with crystal violet (controls were treated in parallel). Representative crystal violet stained colonies are shown in (B), and the mean percent surviving colonies relative to the number of cells plated (± SEM) of 3 triplicate cultures is shown in (C). Mean cell counts ± SEM are listed for each cell line and treatment in Table 2. (*P < .001, **P ≤ .005, 2-sample Student's t-test).