radiation-induced expansion of glioma-initiating cells and resistance

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Research Article

Importance of PKCd signaling in fractionatedradiation-induced expansion of glioma-initiating cells and resistance to cancer treatment Min-Jung Kim1,2, Rae-Kwon Kim1, Chang-Hwan Yoon1, Sungkwan An3, Sang-Gu Hwang4, Yongjoon Suh1, Myung-Jin Park4, Hee Young Chung5, In Gyu Kim2 and Su-Jae Lee1,* 1

Department of Chemistry, Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Korea Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon 305-600, Republic of Korea Functional Genoproteome Research Centre, Konkuk University, Seoul 143-701, Korea 4 Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea 5 Department of Microbiology, College of Medicine, Hanyang University, Seoul 133-791, Korea 2 3

*Author for correspondence ([email protected])

Journal of Cell Science

Accepted 21 May 2011 Journal of Cell Science 124, 3084–3094 ß 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.080119

Summary Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cd (PKCd) is crucial for fractionated-radiation-induced expansion of gliomainitiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCd activity, expanded the CD133-positive (CD133+) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCd expression blocked fractionated-radiation-induced CD133+ cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133+ cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCd activation, suggesting that Abl acts upstream of PKCd. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl–PKCd signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors. Key words: Fractionated radiation, PKCd, Abl, Glioma stem-like cells, Resistance to cancer treatments

Introduction Ionizing radiation is one of the most common and effective treatments for malignant brain tumor (Dewey et al., 1995; Ross, 1999). However, brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation, suggesting that a crucial subpopulation of radiation-resistant tumor cells with potent tumorigenic activity is responsible for tumor regrowth (Dungey et al., 2009; Geoerger et al., 2008; Puchner and Giese, 2000; Welsh et al., 2009). Emerging evidence suggests that such a subpopulation of cancer cells with cancer stem cell properties is responsible for tumor formation, maintenance and progression (Rosen and Jordan, 2009; Singh et al., 2004; Vescovi et al., 2006). These cancer-initiating cells are rare tumor cells characterized by their strong tumorigenic properties and ability to self-renew (Galli et al., 2004; Panchision and McKay, 2002; Singh et al., 2003; Wang et al., 2006). The cancer stem cell population also contributes to resistance to chemotherapy and radiotherapy (Bao et al., 2006; Rich, 2007; Zhang et al., 2010). If a cancer treatment fails to eliminate all self-renewing cancer stem cells, residual surviving cancer stem cells are able to repopulate the tumor, causing

relapse. Furthermore, radiation and chemotherapies used to target gliomas might expand the cancer stem cell population and enhance the aggressiveness of tumors (Campos et al., 2010; Furnari et al., 2007; Mercer et al., 2009; Wang et al., 2010). However, the molecular mechanisms underlying anticancer treatment-induced enrichment of glioma cancer stem cells and the acquisition of resistance have remained unclear. Members of the protein kinase C (PKC) family of serine/ threonine kinases are key components of signal transduction pathways that regulate proliferation, cell survival and malignant transformation (Griner and Kazanietz, 2007; Parekh et al., 2000; Steinberg, 2004). Moreover, there is a substantial body of evidence linking PKC to the initiation and progression of certain types of cancers, including gliomas, and breast, pancreatic and colon cancers (Alonso-Escolano et al., 2006; Bredel and Pollack, 1997; El-Rayes et al., 2008; Gokmen-Polar et al., 2001; Grossoni et al., 2007). Total PKC expression and activity levels are significantly higher in normal brain tissue than in non-brain tissues, suggesting that this serine/threonine kinase has a fundamental role in normal brain physiology (Nelsona and Alkona, 2009; Steinhart et al., 2007). Furthermore, PKC activity

Role of PKCd in cancer progression levels are much higher in brain tumors than in the normal brain. Considerable evidence points to a crucial role for PKC in the malignant phenotype of gliomas. A high level of PKC activity is correlated with rapid proliferation of glioma cells, and inhibitors of PKC greatly reduce glioma proliferation (Graff et al., 2005; Martin and Hussaini, 2005). These and similar observations have led to the concept that inappropriately activated PKC is involved in the development and promotion of cancer; however, the molecular mechanisms by which PKC might contribute to these processes have remained obscure. The goal of this study was to carry out a detailed analysis of the role of the PKC in the acquisition of malignant phenotypes in response to fractionated radiation in human glioma. We show that PKCd activation is essential for the expansion and maintenance of glioma stem-like cell populations and acquisition of resistance to cancer treatments induced by fractionated radiation. The results elucidated in this study provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors. Results


Fractionated radiation induces an increase in the glioma stem-like cell population

Emerging evidence suggests that a CD133-positive (CD133+) cell population possessing tumor-initiating potential is involved in human malignant glioma (Beier et al., 2007), and that the number of CD133+ cells in glioma is increased by cancer treatments (Rosen and Jordan, 2009). To investigate whether fractionated radiation enriched CD133+ glioma stem-like cell populations, we treated two different glioma cell lines, U87 and U373, with 2 Gy of c-radiation per day for 3 days (2 Gy63 days), and analyzed cells for the expression of cancer stem-like cell markers by flow cytometry, polymerase chain reaction (PCR), immunoblotting, and immunostaining. As shown in Fig. 1A, the CD133+ cell populations were significantly expanded in U87 and U373 glioma cells treated with fractionated radiation. Fractionated-radiation treatment also induced an increase in the expression of CD133, nestin and musashi-1, markers of glioma stem-like cells, at both the mRNA and protein levels (Fig. 1B and supplementary material Fig. S1A). Treatment of cells with 6 Gy in a single fraction induced only a slight increase in the size of the CD133+ population (Fig. 1A) and the expression of glioma stem-like cell markers (Fig. 1B and supplementary material Fig. S1A); a single 2 Gy treatment did not cause a significant change in the number of CD133+ cells or the expression levels of marker proteins (data not shown). Moreover, a fluorescence microscopic analysis of cells immunostained with antibodies specific for marker proteins clearly revealed that fractionated radiation increased the expression of CD133, nestin and musashi-1 (Fig. 1C). To determine whether fractionated radiation induces expansion of glioma stem-like cell populations in vivo, we transplanted U87 glioma cells (56106) into athymic nude mice by subcutaneous inoculation, and treated tumors with fractionated radiation (2 Gy for 3 days). Focal treatment of tumors with fractionated radiation significantly expanded the CD133+ cell population (Fig. 1D), and also induced the expression of CD133, nestin and musashi-1 (Fig. 1E). These results indicate that fractionated radiation effectively induces expansion of glioma stem-like cell populations in vivo. To further determine whether fractionated radiation influences self-renewal of glioma stem-like cells, we examined changes in the expression levels of the known self-renewal-related proteins Sox2, Notch1/2 and b-catenin. As

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shown in Fig. 1F and supplementary material Fig. S1B, Sox2, Notch2 and b-catenin were markedly upregulated in glioma cells treated with fractionated radiation, whereas the levels of Notch1 were not changed. These results suggest that fractionated radiation might enhance self-renewal ability in glioma stem-like cells. Fractionated radiation causes a decrease in cellular sensitivity to chemotherapeutic agents

We next examined changes in cellular sensitivity to chemotherapeutic agents in glioma cells treated with fractionated radiation. In these experiments, we first exposed U87 and U373 glioma cells to fractionated radiation, then next day cells were replated and cultured for another day. Cells were then treated with cisplatin, etoposide or paclitaxel for 48 hours and cell death was analyzed by flow cytometry. Interestingly, pretreatment of glioma cells with fractionated radiation significantly decreased the sensitivity of both glioma cell lines to the three different chemotherapeutic treatments (Fig. 2 and supplementary material Fig. S2). These results suggest that fractionated radiation induces an increase in the resistance of human glioma cells to anticancer therapeutics. PKCd activation is required for fractionated-radiationinduced enrichment of glioma stem-like cells

PKCs have been implicated in the progression and maintenance of the malignant phenotype of certain cancers, including gliomas, and colon, pancreatic and breast cancers (Alonso-Escolano et al., 2006; Bredel and Pollack, 1997; El-Rayes et al., 2008; Gokmen-Polar et al., 2001; Grossoni et al., 2007). Potentiation of the malignant phenotype might be mediated by activation of selective PKC isoenzymes or through altered expression of the isoenzyme profile compared with the originating tissue (Steinberg, 2004). To determine whether PKC is involved in the fractionated-radiationinduced enrichment of glioma stem-like cells, we first examined the activation status of PKC isoforms using an immune complex kinase assay. As shown in Fig. 3A, PKCb and PKCd activities were selectively increased in glioma cells after treatment with fractionated radiation. The activity of PKCa and PKCf was not altered, and there were no changes in any PKC isoforms at the total protein level. To further determine whether PKCb and/or PKCd activity are required for the fractionated-radiation-induced enrichment of glioma stem-like cell populations, we treated cells with inhibitors of specific PKC isoforms and analyzed changes in the CD133+ cell population and the expression of glioma stem-like cell marker proteins. As shown in Fig. 3B, treatment with the broad-spectrum PKC inhibitor Go¨6850 markedly suppressed induction of the CD133+ cell population by fractionated radiation. Importantly, fractionated-radiation-induced expansion of the CD133+ cell population was clearly suppressed by treatment with the specific PKCd inhibitor Rottlerin (Fig. 3B). The expression of glioma stem cell marker proteins nestin and musashi-1 was also reduced by Go¨6850 and Rottlerin (Fig. 3C and supplementary material Fig. S3A). By contrast, Go¨6976, a specific inhibitor of PKCa and PKCb, failed to inhibit fractionatedradiation-induced enrichment of the CD133+ cell population (Fig. 3B) and expression of glioma stem cell marker proteins (Fig. 3C and supplementary material Fig. S3A). We next attempted to reproduce the effects seen with PKC inhibitors by performing single and combinational knockdown experiments with smallinterfering RNAs (siRNAs) against individual PKC isoforms. Consistent with the results obtained using pharmacological

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Journal of Cell Science 124 (18)


inhibitors, siRNA against PKCd selectively attenuated expansion of the CD133+ cell population (Fig. 3D), but siRNAs against other PKC isozymes (PKCa, PKCb and PKCf) did not (Fig. 3D). To control for potential off-target effects of individual PKCd-specific siRNAs, we used three different siRNAs against PKCd. Each of

Fig. 1. See next page for legend.

these different siRNAs clearly attenuated fractionated-radiationinduced expansion of the CD133+ cell population (Fig. 3E). Moreover, flow cytometry analyses, immunoblotting and immunocytochemistry revealed that PKCd-specific siRNA suppressed expression of CD133, nestin and musashi-1


Role of PKCd in cancer progression

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Fig. 2. Acquisition of resistance to chemotherapeutic agents by fractionated radiation. U87 and U373 cells were exposed to fractionated radiation and then treated with cisplatin, etoposide or paclitaxel. After 48 hours, cells were harvested, fixed and stained with propidium iodide (PI) and FACS analyses were performed. The percentage of sub-G1 (apoptotic) cells is indicated in each experiment. The data represent means s.d. from three experiments (P,0.01).

(Fig. 3F,G and supplementary material Fig. S3B). Importantly, siRNA targeting PKCd effectively inhibited expression of the known self-renewal-related proteins Sox2, Notch2 and b-catenin in cells treated with fractionated radiation (Fig. 3H and supplementary material Fig. S3C). These results specifically implicate PKCd in the expansion and maintenance of glioma stem-like cell populations by fractionated radiation. Fig. 1. Expansion of glioma stem-like cell populations by fractionated radiation. (A) U87 and U373 cells were exposed to fractionated radiation (2 Gy63) or a single radiation dose (6 Gy); 48 hours later, cells were analyzed for expression of the cancer stem-like cell marker CD133 by flow cytometry. The data represent means ¡ s.d. from six experiments (P,0.01). (B) Left: expression level of mRNA encoding CD133, nestin and musashi-1 was examined by RT-PCR. GAPDH served as an internal standard. Right: whole-cell lysates were analyzed by immunoblotting using antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (See supplementary material Fig. S1 for a densitometric analysis.) (C) U87 cells were immunostained with antibodies against CD133, nestin or musashi-1 and imaged by fluorescence microscopy. (D) U87 cells were harvested, counted and suspended in an equal volume of high-concentration Matrigel; 100 ml of the suspension (56106 cells) was injected subcutaneously into the right flank of nude mice. After allowing the uniform development of visible tumors, mice were exposed to focal radiation (2 Gy of c-radiation on three consecutive days). After 48 hours, the tumors were surgically removed, dissociated to single cells and then analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (E) Cell lysates were subjected to immunoblot analysis with antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (F) Whole-cell lysates were subjected to immunoblot analysis with antibodies against Sox2, Oct4, Notch1/2 and b-catenin. b-actin was used as a loading control. (See supplementary material Fig. S1 for a densitometric analysis.)

PKCd activation is required for the acquisition of resistance to chemotherapeutic agents

To examine whether activation of PKCd is involved in the acquisition of resistance to chemotherapeutics caused by fractionated radiation, we specifically inhibited PKCd activity with Rottlerin or knocked down PKCd expression with a specific siRNA. As shown in Fig. 4A, inhibition of PKCd activity with Rottlerin significantly sensitized to cisplatin, etoposide and paclitaxel chemotherapeutic agents in glioma cells pretreated with fractionated radiation leading to more cell death, compared with non-treated cells. However, the PKCa and PKCb inhibitor Go¨6976 had no effect (data not shown). siRNA-mediated knockdown of PKCd also clearly reduced the resistance of fractionated-radiationtreated glioma cells to all three cancer treatments (Fig. 4B). Furthermore, pretreatment with shRNA targeted to PKCd before fractionated radiation decreased clonogenic survival to the cancer drugs, cisplatin, etoposide and paclitaxel, compared with fractionated-radiation treatment alone or non-treated glioma cells within a range of concentration of cancer drugs that we tested (Fig. 4C). These results indicate that the PKCd signaling pathway is involved in the acquisition of resistance to chemotherapeutic agents in human glioma cells exposed to fractionated radiation. Activation of Abl is involved in PKCd-mediated enrichment of glioma stem-like cells and resistance to cancer treatment

Non-receptor tyrosine kinases, including Abl and Src (Src tyrosine kinase), are well-known upstream activators of PKC in response to genotoxic and oxidative stress (Choi et al., 2006; Rodriguez et al., 2009), and have a role in cancer progression (Parekh et al., 2000). To investigate the potential involvement of non-receptor tyrosine



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Fig. 3. See next page for legend.


Role of PKCd in cancer progression

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Fig. 4. Inhibition of PKCd restores fractionated-radiation-induced resistance of glioma cells to chemotherapeutic agents. (A) U87 cells were treated with the PKCd inhibitor Rottlerin and then exposed to fractionated radiation. Cells were then treated with cisplatin (50 mM), etoposide (12.5 mM) or paclitaxel (250 nM); after 48 hours, apoptotic cell death was assessed by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (B) U87 cells were transfected with siRNA against PKCd or with control siRNA and then exposed to fractionated radiation. Cells were then treated with cisplatin, etoposide or paclitaxel; after 48 hours, apoptotic cell death was assessed by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (C) Cell survival was analyzed by counting colony formation. U87 cells were infected with shRNA against PKCd or control and then exposed with fractionated radiation. Cells were treated with cisplatin, etoposide or paclitaxel. After 7 days of incubation, colonise were stained with Trypan Blue dye and the number of colonies was counted. The data represent means ¡ s.d. from three experiments (P,0.01).

kinases in the fractionated-radiation-induced enrichment of glioma stem-like cell populations and acquisition of resistance to anticancer treatment, we performed immune-complex kinase assays using antibodies against Src and Abl kinases. As shown in Fig. 5A, Abl kinase activity was markedly increased in cells

treated with fractionated radiation without a corresponding change in Abl protein expression levels. siRNA-mediated knockdown of Abl led to a significant decrease in the CD133+ cell population (Fig. 5B) and suppressed expression of the marker proteins CD133, nestin and musashi-1 (Fig. 5C). Moreover, Abl RNAi

Fig. 3. Role of PKCd in fractionated-radiation-induced expansion of glioma stem-like cells. (A) U87 cells were exposed to fractionated radiation or a single radiation dose. After 48 hours, cells were analyzed for kinase activities of PKC isotypes using immune complex kinase assays. (B) U87 cells were treated with the PKC inhibitors Go¨6850, Go¨6976 or Rottlerin and then exposed to fractionated radiation. After 48 hours, cells were analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (C) Whole-cell lysates were subjected to immunoblot analysis with antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (See supplementary material Fig. S3A for a densitometric analysis.) (D) U87 cells were transfected with siRNA against PKCa, PKCb, PKCd or PKCf, or with control siRNA and then exposed to fractionated radiation. After 48 hours, cells were analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (E) U87 cells were transfected with siRNA against PKCd-1, PKCd-2 or PKCd-3, or with control siRNA and then exposed to fractionated radiation. After 48 hours, cells were analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (F) Whole-cell lysates were subjected to immunoblot analysis with antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (See supplementary material Fig. S3B for a densitometric analysis.) (G) Cells were immunostained with antibodies against CD133, nestin or musashi-1 and imaged by fluorescence microscopy. (H) Whole-cell lysates were subjected to immunoblot analysis with antibodies against Sox2, Notch1/2 and b-catenin antibodies. b-actin was used as a loading control. (See supplementary material Fig. S3 for a densitometric analysis.)

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Journal of Cell Science 124 (18) fractionated radiation (Fig. 5A), but knockdown of Src with siRNA did not alter the CD133+ cell population (Fig. 5B) or expression of marker proteins (data not shown). These results indicate that Abl acts upstream of PKCd activation in response to fractionated radiation, and has a role in enrichment of the glioma stem-like cell population and acquisition of cancer drug resistance.


effectively inhibited PKCd activation by fractionated radiation (Fig. 5D) and significantly restored cellular sensitivity to chemotherapeutic agents (Fig. 5E). In addition, combined Abl knockdown and PKCd overexpression rescued the resistance to cancer treatment induced in glioma cells by fractionated radiation (Fig. 5F). Src kinase activity was also slightly increased by

Fig. 5. Role of Abl in fractionated-radiation-induced expansion of glioma stem-like cells. (A) U87 cells were exposed to fractionated radiation; after 48 hours, cells were analyzed for Abl and Src kinase activity by immune complex kinase assay. (B) U87 cells were transfected with siRNA against Abl or Src, or with control siRNA and then exposed to fractionated radiation. After 48 hours, cells were analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (C) Whole-cell lysates were subjected to immunoblot analysis with antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (D) Cells were analyzed for PKCd kinase activity using an immune complex kinase assay. (E) Cells were treated with cisplatin, etoposide, or paclitaxel; after 48 hours, apoptotic cell death was assessed by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (F) U87 cells were transfected with wild-type PKCd and/or siRNA against Abl. Cells were then exposed to fractionated radiation and treated with cisplatin, etoposide or paclitaxel. Apoptotic cell death was assessed by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01).

Role of PKCd in cancer progression PKCd is crucial for the fractionated-radiation-induced expansion of glioma-initiating cells and resistance to cancer treatment in patient-derived glioma cells


To further confirm the role of PKCd in the fractionated-radiationinduced expansion of glioma stem-like cell populations, we isolated glioma cells from two different patients (X01GB and X03AOA), treated these cells with fractionated radiation, and examined changes in the CD133+ cell population, expression of gliomainitiating-cell marker proteins, and cellular sensitivity to cisplatin, etoposide and paclitaxel after knocking down PKCd activity with a specific siRNA. As shown in Fig. 6A, siRNA-mediated PKCd knockdown clearly attenuated fractionated radiation-induced enrichment of the CD133+ cell population in glioma cells derived from both patients. The expression of glioma stem-like cell markers nestin and musashi-1 was also inhibited by siRNAs targeting PKCd (Fig. 6B). Moreover, downregulation of PKCd reduced the resistance to cancer treatment induced in patient-derived glioma cells by fractionated radiation (Fig. 6C). These results suggest that activation of PKCd is crucial for the fractionated-radiation-induced expansion of glioma stem-like cell populations and acquisition of resistance to chemotherapeutic agents in human glioma cells. Discussion Radiation therapy is a very effective treatment that is used to help destroy malignant brain tumors. However, brain tumors frequently recur or progress after radiation treatment, suggesting that a crucial subcellular population of radiation-resistant tumor cells with potent tumorigenic activity is responsible for re-growth. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained obscure. In this study, we demonstrate

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that PKCd has a crucial role in fractionated-radiation-induced expansion of glioma stem-like cell population and acquisition of resistance to chemotherapeutic treatment. A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance and progression (Galli et al., 2004; Rosen and Jordan, 2009; Singh et al., 2004; Vescovi et al., 2006); contributes to resistance to chemotherapy and radiotherapy (Bao et al., 2006; Rich, 2007; Zhang et al., 2010); and might be expanded by radiation- and chemotherapy used to target gliomas, thereby making tumors more aggressive (Campos et al., 2010; Furnari et al., 2007; Mercer et al., 2009; Wang et al., 2010). In this study, we also found that treatment of glioma cells with fractionated radiation (2 Gy for 3 days) expanded the subpopulation of cells with tumor-initiating potential. Fractionated radiation also increased expression levels of the cancer stem-like cell markers CD133, nestin and musashi-1 in glioma cells. Moreover, the expression levels of the self-renewal related proteins Sox2 and Notch2 were also markedly upregulated in glioma cells treated with fractionated radiation. We additionally found that cells treated with fractionated radiation were less sensitive to the chemotherapeutic agents cisplatin, etoposide and paclitaxel. These results suggest that fractionated radiation causes an increase in glioma cancer stem-like cell populations, and also increases the resistance of human glioma cells to anticancer treatments. Members of the PKC family of serine/threonine kinases are key components of signal transduction pathways that have been linked to carcinogenesis and cancer progression (Griner and Kazanietz, 2007; Steinberg, 2004). PKC has also been implicated in the acquisition of resistance to anticancer treatments in malignant tumors (Bredel, 2001). Here, we found that

Fig. 6. Role of PKCd in the fractionated-radiation-induced expansion of glioma-stem-like cells in patient-derived glioma cells. (A) Glioma cells derived from two patients (X01GB and X03AOA) were transfected with siRNA against PKCd or with control siRNA, and then exposed to fractionated radiation. After 48 hours, cells were analyzed for CD133 expression by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01). (B) Whole-cell lysates were subjected to immunoblot analysis with antibodies against CD133, nestin and musashi-1. b-actin was used as the loading control. (C) Cells were treated with cisplatin, etoposide or paclitaxel; after 48 hours, apoptotic cell death was assessed by flow cytometry. The data represent means ¡ s.d. from three experiments (P,0.01).


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fractionated radiation caused a marked increase in activity of PKCd. Importantly, inhibition of PKCd led to a decrease in the glioma stem-like cell population, and clearly restored cellular sensitivity to chemotherapeutic agents in cells treated with fractionated radiation. In previous studies, PKC was shown to have a role in the maintenance of the malignant phenotype of gliomas (Cheng et al., 2010; da Rocha et al., 2002). High levels of PKC activity are correlated with rapid proliferation of glioma cells; conversely, inhibition of PKC reduces glioma proliferation (da Rocha et al., 2002). These observations, taken together with our findings, support the idea that inappropriately activated PKC is involved in the promotion of cancer, especially brain tumor, and reinforce the suggestion that selective inhibitors of PKC could have wide-ranging therapeutic potential. Previous reports demonstrated that, in response to diverse stimuli, Src kinase promotes activation of PKCd (Rodriguez et al., 2009). We also found that fractionated radiation induced a slight activation of Src kinase in glioma cells. However, inhibition of Src with a specific siRNA did not affect fractionated-radiation-induced PKCd activation or enrichment of the CD133+ cell population. Instead, we found that fractionated-radiation-induced PKCd activation was associated with Abl tyrosine kinase. It has been suggested that Abl is activated in response to genotoxic and oxidative stress, and activated Abl interacts with PKCd in response to both stimuli to activate PKCd (Choi et al., 2006). We also found that Abl directly interacted with PKCd in response to fractionated radiation (data not shown). Moreover, siRNA targeting of Abl attenuated fractionated-radiation-induced enrichment of the glioma-initiating cell population and effectively restored chemotherapeutic sensitivity, suggesting that Abl is located upstream of PKCd activation in the response to fractionated radiation, and that it has a role in the enrichment of the glioma stem-like cell population and the acquisition of anticancer drug resistance. Collectively, our findings show that fractionated radiation causes an increase in the glioma stem-like cell population and a decrease in cellular sensitivity to chemotherapeutic agents. Importantly, we found that activation of Abl–PKCd signaling is critically required for both these fractionated-radiation-induced events. These findings provide insights that might prove pivotal in the context of radiation-based therapeutic intervention for brain tumors. Materials and Methods Chemical reagents and antibodies

Go¨6850, Go¨6976, Rottlerin, cisplatin and etoposide were purchased from Calbiochem (San Diego, CA). Polyclonal antibodies against PKCa, PKCb, PKCd, PKCf, Abl and Src were purchased from Santa Cruz (Santa Cruz, CA). Polyclonal antibodies against CD133, nestin, musashi-1 and Notch2 were obtained from Abcam (San Diego, CA). Polyclonal anti-Notch1 was obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-b-catenin antibody was obtained from BD Biosciences. Monoclonal anti-b-actin antibody and paclitaxel were obtained from Sigma (St Louis, MO).

Irradiation

Cells were plated in 60 mm dishes and irradiated at room temperature with a 137Cs laboratory c-irradiator (AtomicEnergy of Canada, Ltd, Mississauga, Canada) at a dose rate of 3.81 Gy/minute for the time required to apply a prescribed dose. For fractionated radiation of 2 Gy, cells were irradiated on three consecutive days. The single dose radiation was applied at the same time as the last dose of fractionated radiation. Cells were harvested 48 hours after the last fraction of radiation. siRNA transfection

Double-stranded siRNAs targeting PKCa (GenBank acc. no. BC109274), PKCb (BC109274), PKCd (BC043350), PKCf (BC014270), Abl (BC117451) and Src (BC104875) were synthesized by Ambion (Austin, TX). All siRNAs were 21 nucleotides long and contained symmetric 39 overhangs of two deoxythymidines. Sequences are as follows: PKCa, 59-GGAGCACAGGGUUCGUAGATT-39 and 59-GGAUUGUCUACGAACCCUGUUATT-39; PKCb, 59-GCCAGUGUUGAUGGCUGGUTT-39 and 59-ACCAGCCAUCAACACUGGCTT-39; PKCd-1, 59-GCUUCAAGGUUCACAACUATT-39 and 59-UAGUUGUGAACCUUGAAGCTT-39; PKCd-2, 59-GCCCACCAUGUACCCUGAGTT-39 and 59-AGUGGGCGUCGAAUGTGGATT-39; PKCd-3, 59-CGAGAAGAUCAUCGGCAGATT-39 and 59-UCUGCCGAUGAUCUUGUCGTT-39; PKCf, 59-GUGUCCUUAUGUUUGAGAUTT-39 and 59-AUCUCAAACAUAAGGACACTT-39; Abl, 59-GCAGAGUUCAAAAGCCCUUTT-39 and 59-AGCAGAGUUCAAAAGCCCUTT-39; Src, 59-AACAAGAGCAAGCCCAAGGTT-39 and 59-AAGCACUACAAGAUCCGCATT-39; scrambled siRNA, 59-AATTCTCACACTTCGGAGATT-39 and 59-AAGTTCTCCGAAGTGTGAGTT-39. All template oligonucleotides siRNA duplexes (50 nM) were introduced into cells using a Neon Transfection System (Invitrogen) by following the procedure recommended by the manufacturer. To monitor the effect of siRNA, we performed immunoblot analysis at 48 hours after transfection. Lentiviral shRNA experiments

MISSION short-hairpin RNA (shRNA) plasmids (Sigma) encoding siRNAs targeting PKCd or control were purchased from Sigma. Plasmids (PKCd: TRCN0000010193, control: SHC202V) were effective in knocking down PKCd expression. To standardize lentiviral transduction assays, viral titers were measured in a U87 cell line. For growth assays, titers corresponding to multiplicities of infection (MOIs) of 5 and 1 in U87 cells were used. For PKCd knockdown, cells were plated on day zero at 16105 cells in 60 mm dish. Cells were infected and after 24 hours, cells were treated with 1 mg/ml puromycin for 3 days to eliminate uninfected cells. Medium was replaced, and after 2 more days, cells were harvested for western blot analysis (Singh et al., 2009; Moffat et al., 2006; Naldini et al., 1996). Fluorescence microscopy

56104 cells were plated on glass slides and fixed with 4% paraformaldehyde. Following cell fixation, cells were incubated with the appropriate primary antibodies in a solution of PBS with 1% bovine serum albumin and 0.1% Triton X100 at 4 ˚C overnight. Antibodies against human proteins were as follows: CD133 (rabbit polyclonal antibody, 1:200), nestin (rabbit polyclonal antibody, 1:200) and musashi-1 (rabbit polyclonal antibody, 1:200) for the detection of neural stem and progenitor cells. Staining was visualized using anti-rabbit Alexa Flour 488 (Molecular Probes). Nuclei were counterstained using 4,6-diamidino-2phenylindole (DAPI; Sigma). Stained cells were visualized with a fluorescence microscope (Olympus IX71). Quantification of apoptotic cell death

Cells were collected and washed with PBS. After fixation with 70% ethanol, cells were washed twice with PBS and stained with a solution containing 20 mg/ml propidium iodide and 50 mg/ml RNase A. Cells were incubated for 30 minutes at the room temperature and cell cycle profiles were determined by flow cytometry using a FACScan (Becton-Dickinson, San Jose, CA). The percentage of cells at sub-G1 (apoptotic cells) is indicated in each experiment. Clonogenic survival assay

Cells were plated in triplicate into 60 mm culture dishes at a density of 500 colonies per dish. After 7 days of incubation, the culture medium was decanted and the colonies were fixed with a mixture of 75% methanol and 25% acetic acid. Colonies were stained with 0.4% Trypan Blue dye and the number of colonies .5 mm was counted.

Cell culture and patient-derived glioma cells

U87 and U373 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM medium supplemented with 10% heatinactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere. Patient-derived glioma cells maintained in DMEM medium supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere.

Western blot analysis

Cell lysates were prepared by extracting proteins with lysis buffer (40 mM TrisHCl, pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline and incubated with primary antibodies for overnight at 4 ˚C. Blots were developed with a peroxidase-

Role of PKCd in cancer progression conjugated secondary antibody and proteins visualized by enhanced chemiluminescence (ECL) procedures (Amersham, Arlington Heights, IL), using the manufacturer’s protocol. Immune complex kinase assay

Proteins from 300 mg of cell extracts were immunoprecipitated with primary antibody (2 mg/ml) at 4 ˚C for 4 hours. The immunoprecipitates were washed twice in kinase reaction buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 2.5 mM EGTA, 1 mM NaF, 0.1 mM Na3VO4 and 10 mM bglycerophosphate) and then resuspended in 20 ml of kinase reaction buffer. The kinase assay was initiated by adding 20 ml of kinase reaction buffer, containing 10 g of substrate and 2 mCi of [c-32P]ATP. The reactions were carried out at 30 ˚C for 30 minutes and terminated by adding SDS sample buffer and the mixtures were boiled for 5 minutes. The reaction products were analyzed by SDS-PAGE and autoradiography. RNA isolation and RT-PCR

Total mRNA was isolated using the RNeasy kit (Qiagen, Valencia, CA). RT-PCR reactions were performed with SuperScript III (Invitrogen) according to the manufacturer’s instructions. The expression of autophagy genes was determined by RT-PCR using the following primers: CD133, 59-GACAAAGATGTGCTTCGAGATGTG-39 and 59-GTAGCTCAGATGCTCGCTCAG-39; nestin, 59-CTGAAACTGGACACGAGCTTCAAG-39 and 59-CCAGAACAGTATAACGGCAACTCC-39; musashi-1, 59-CAAAGCCTCCAAAATTCAGC-39 and 59-GAAGCAGAAAGGCAGCATA-39; GAPDH, 59-CCATGGAGAAGGCTGGGG-39 and 59-CAAAGTTGTCATGGATGACC-39.


Flow cytometry

Cells were trypsinized and pretreated with FcR-blocking reagent, and then incubated in human anti-CD133/1-PE antibody (Miltenyi Biotech) or isotype control antibody (mIgG2b-PE, Miltenyi Biotech) for 30 minutes at 4 ˚C. Cells were subjected to flow cytometry analysis using a FACSCalibur cytometer (Becton Dickinson). Three independent experiments were performed. Solid tumor xenografts in nude mice

All animal procedures and care were approved by the Korea Institute of Radiological and Medical Sciences. Tumors were formed on the right flank by subcutaneous inoculation of 56106 U87 cells into athymic Balb/c female nude mice (5 weeks of age; Charles River Laboratories). When tumors grew to 400 mm3 in diameter, animals were divided into two groups of ten mice each. Mice were exposed to focal radiation (2 Gy of c-radiation on three consecutive days). Tumor volume was calculated using the formula [(smallest diameter2 6 widest diameter)/ 2]. The solid tumors were surgically removed and the tumors were photographed. Solid tumor tissues were finely minced using a razor blade, washed in DMEM and then incubated with Accumax 1 for 10 minutes at 37 ˚C. Single-cell suspension was obtained by filtering digested tissue through a 70 m cell strainer and then gently loaded onto a layer of Histopaque-1077 gradient (Sigma). After centrifugation at 400 g for 30 minutes at room temperature, red blood cells, dead cells and debris were removed from the bottom of the tube and live nucleated cells were collected at the interface. Cells were analyzed for the expression of cancer-initiating cell marker CD133 by flow cytometry and subjected to immunoblot analysis. Statistical analysis

All the data in the study were performed in triplicate. The results are described as mean ¡ s.d. Statistical analysis was performed by one-way analysis of variance (ANOVA) and comparisons among groups were performed by independent sample t-test. Differences with P,0.01 were considered statistically significant.

This work was supported by the Korea Research Foundation (KRF) and Ministry of Education, Science and Technology (MEST), Korean government, through its National Nuclear Technology Program (20082003935) and Medical Research Center Program (2010-0091464). Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.080119/-/DC1

References Alonso-Escolano, D., Medina, C., Cieslik, K., Radomski, A., Jurasz, P., SantosMartinez, M. J., Jiffar, T., Ruvolo, P. and Radomski, M. W. (2006). Protein kinase C delta mediates platelet-induced breast cancer cell invasion. J. Pharmacol. Exp. Ther. 318, 373-380. Bao, S., Wu, Q., McLendon, R. E., Hao, Y., Shi, Q., Hjelmeland, A. B., Dewhirst, M. W., Bigner, D. D. and Rich, J. N. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760.

3093

Beier, D., Hau, P., Proescholdt, M., Lohmeier, A., Wischhusen, J., Oefner, P. J., Aigner, L., Brawanski, A., Bogdahn, U. and Beier, C. P. (2007). CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res. 67, 4010-4015. Bredel, M. (2001). Anticancer drug resistance in primary human brain tumors. Brain Res. Brain Res. Rev. 35, 161-204. Bredel, M. and Pollack, I. F. (1997). The role of protein kinase C (PKC) in the evolution and proliferation of malignant gliomas, and the application of PKC inhibition as a novel approach to anti-glioma therapy. Acta Neurochir. 139, 1000-1013. Campos, B., Wan, F., Farhadi, M., Ernst, A., Zeppernick, F., Tagscherer, K. E., Ahmadi, R., Lohr, J., Dictus, C., Gdynia, G. et al. (2010). Differentiation therapy exerts antitumor effects on stem-like glioma cells. Clin. Cancer Res. 16, 2715-2728. Cheng, L., Bao, S. and Rich, J. N. (2010). Potential therapeutic implications of cancer stem cells in glioblastoma. Biochem. Pharmacol. 80, 654-665. Choi, S. Y., Kim, M. J., Kang, C. M., Bae, S., Cho, C. K., Soh, J. W., Kim, J. H., Kang, S., Chung, H. Y., Lee, Y. S. et al. (2006). Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells. J. Biol. Chem. 281, 7049-7059. da Rocha, A. B., Mans, D. R., Regner, A. and Schwartsmann, G. (2002). Targeting protein kinase C: new therapeutic opportunities against high-grade malignant gliomas? Oncologist 7, 17-33. Dewey, W. C., Ling, C. C. and Meyn, R. E. (1995). Radiation-induced apoptosis: relevance to radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 33, 781-796. Dungey, F. A., Caldecott, K. W. and Chalmers, A. J. (2009). Enhanced radiosensitization of human glioma cells by combining inhibition of poly(ADPribose) polymerase with inhibition of heat shock protein 90. Mol. Cancer Ther. 8, 2243-2254. El-Rayes, B. F., Ali, S., Philip, P. A. and Sarkar, F. H. (2008). Protein kinase C: a target for therapy in pancreatic cancer. Pancreas 36, 346-352. Furnari, F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J. M., Stegh, A., Hahn, W. C., Ligon, K. L., Louis, D. N., Brennan, C. et al. (2007). Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 21, 2683-2710. Galli, R., Binda, E., Orfanelli, U., Cipelletti, B., Gritti, A., De Vitis, S., Fiocco, R., Foroni, C., Dimeco, F. and Vescovi, A. (2004). Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 64, 7011-7021. Geoerger, B., Gaspar, N., Opolon, P., Morizet, J., Devanz, P., Lecluse, Y., Valent, A., Lacroix, L., Grill, J. and Vassal, G. (2008). EGFR tyrosine kinase inhibition radiosensitizes and induces apoptosis in malignant glioma and childhood ependymoma xenografts. Int. J. Cancer 123, 209-216. Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z. and Fields, A. P. (2001). Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis. Cancer Res. 61, 1375-1381. Graff, J. R., McNulty, A. M., Hanna, K. R., Konicek, B. W., Lynch, R. L., Bailey, S. N., Banks, C., Capen, A., Goode, R., Lewis, J. E. et al. (2005). The protein kinase Cbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res. 65, 7462-7469. Griner, E. M. and Kazanietz, M. G. (2007). Protein kinase C and other diacylglycerol effectors in cancer. Nat. Rev. Cancer 7, 281-294. Grossoni, V. C., Falbo, K. B., Kazanietz, M. G., de Kier Joffe, E. D. and Urtreger, A. J. (2007). Protein kinase C delta enhances proliferation and survival of murine mammary cells. Mol. Carcinog. 46, 381-390. Martin, P. M. and Hussaini, I. M. (2005). PKC eta as a therapeutic target in glioblastoma multiforme. Expert Opin. Ther. Targets 9, 299-313. Mercer, R. W., Tyler, M. A., Ulasov, I. V. and Lesniak, M. S. (2009). Targeted therapies for malignant glioma: progress and potential. BioDrugs 23, 25-35. Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K. et al. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283-1298. Naldini, L., Blo¨mer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. and Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267. Nelsona, T. J. and Alkona, D. L. (2009). Neuroprotective versus tumorigenic protein kinase C activators. Trends Biochem. Sci. 34, 136-145. Panchision, D. M. and McKay, R. D. (2002). The control of neural stem cells by morphogenic signals. Curr. Opin. Genet. Dev. 12, 478-487. Parekh, D. B., Ziegler, W. and Parker, P. J. (2000). Multiple pathways control protein kinase C phosphorylation. EMBO J. 19, 496-503. Puchner, M. J. and Giese, A. (2000). Tamoxifen-resistant glioma-cell sub-populations are characterized by increased migration and proliferation. Int. J. Cancer 86, 468-473. Rich, J. N. (2007). Cancer stem cells in radiation resistance. Cancer Res. 67, 8980-8984. Rodriguez, E. M., Dunham, E. E. and Martin, G. S. (2009). Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Srctransformed cells. J. Cell. Physiol. 221, 171-182. Rosen, J. M. and Jordan, C. T. (2009). The increasing complexity of the cancer stem cell paradigm. Science 324, 1670-1673. Ross, G. M. (1999). Induction of cell death by radiotherapy. Endocr. Relat. Cancer 6, 41-44. Singh, A., Greninger, P., Rhodes, D., Koopman, L., Violette, S., Bardeesy, N. and Settleman, J. (2009). A gene expression signature associated with ‘‘K-Ras

3094



addiction’’ reveals regulators of EMT and tumor cell survival. Cancer Cell 15, 489500. Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J. and Dirks, P. B. (2003). Identification of a cancer stem cell in human brain tumors. Cancer Res. 63, 5821-5828. Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D. and Dirks, P. B. (2004). Identification of human brain tumour initiating cells. Nature 432, 396-401. Steinberg, S. F. (2004). Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem. J. 384, 449-459. Steinhart, R., Kazimirsky, G., Okhrimenk, H., Ben-Hur, T. and Brodie, C. (2007). PKCepsilon induces astrocytic differentiation of multipotential neural precursor cells. Glia 55, 224-232.

Vescovi, A. L., Galli, R. and Reynolds, B. A. (2006). Brain tumour stem cells. Nat. Rev. Cancer 6, 425-436. Wang, J., Wakeman, T. P., Lathia, J. D., Hjelmeland, A. B., Wang, X. F., White, R. R., Rich, J. N. and Sullenger, B. A. (2010). Notch promotes radioresistance of glioma stem cells. Stem Cells 28, 17-28. Wang, S., Garcia, A. J., Wu, M., Lawson, D. A., Witte, O. N. and Wu, H. (2006). Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc. Natl. Acad. Sci. USA 103, 1480-1485. Welsh, J. W., Mahadevan, D., Ellsworth, R., Cooke, L., Bearss, D. and Stea, B. (2009). The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells. Radiat. Oncol. 4, 69. Zhang, M., Atkinson, R. L. and Rosen, J. M. (2010). Selective targeting of radiationresistant tumor-initiating cells. Proc. Natl. Acad. Sci. USA 107, 3522-3527.