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Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance
FEBS Letters 586 (2012) 3429–3434
journal homepage: www.FEBSLetters.org
Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance NUPR1 and p21 in chemoresistance Andrew J. Vincent a, Suping Ren a, Lillianne G. Harris a, Daniel J. Devine a, Rajeev S. Samant b,c, Oystein Fodstad d,e, Lalita A. Shevde b,c,⇑ a
Mitchell Cancer Institute, University of South Alabama, Mobile, AL, United States Department of Pathology, University of Alabama at Birmingham, United States Comprehensive Cancer Center, University of Alabama at Birmingham, United States d Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway e Institute for Clinical Medicine, Faculty of Medicine, University in Oslo, Oslo, Norway b c
a r t i c l e
i n f o
Article history: Received 29 March 2012 Revised 9 July 2012 Accepted 22 July 2012 Available online 31 July 2012 Edited by Varda Rotter Keywords: NUPR1 P21 Chemoresistance Akt IKK-b
a b s t r a c t The expression of Nuclear Protein 1 (NUPR1) is associated with chemoresistance in multiple malignancies. We previously reported that NUPR1 functions as a transcriptional cofactor for the p300–p53 complex and transcriptionally regulates p21 expression. In the present study we investigated the activity of NUPR1 in p53-deficient, triple-negative, inflammatory SUM159 breast cancer cells. Our studies reveal that NUPR1 confers growth benefit and chemoresistance by causing Akt-mediated phosphorylation and subsequent cytoplasmic re-localization of p21 and activation of the anti-apoptotic Bcl-xL protein. Our findings elucidate a NUPR1-PI-3-K/Akt-phospho-p21 axis that functions in p53-negative, inflammatory breast cancer cells to enhance chemoresistance in breast cancer. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Much progress has been made in the treatment of breast cancer, but in metastatic disease chemoresistance remains a barrier to long-term survival. A number of clinical trials have demonstrated that complete response occurs in only one-third of patients with recurrent breast cancer [1,2]. Hence, there is an urgent need to identify molecular mechanisms that regulate chemoresistance. Nuclear Protein 1 (NUPR1, also known as com1 and p8) is a small molecule whose levels are upregulated in response to cellular stress and also in multiple malignancies [3–7] and are inversely correlated with apoptosis and autophagy [8]. We and others have shown that NUPR1 confers resistance to multiple drugs, including gemcitabine, paclitaxel and doxorubicin [9]. NUPR1 plays a role in tumor progression and distant metastasis of breast cancer [10] and is shown to regulate many steps in metastasis, including migration, invasion, and adhesion [11]. We have reported that NUPR1 mediates doxorubicin resistance in a p53-dependent manner by transcriptionally upregulating p21 [9]. p21 is a well-known negative regulator of cell cycle progression. ⇑ Corresponding author. Address: Department of Pathology, University of Alabama at Birmingham, 1824 6th Avenue South, Birmingham, AL 35233, United States. Fax: +1 205 975 6615. E-mail address: [email protected] (L.A. Shevde).
Recent studies show that p21 can act both as a tumor suppressor gene and an oncogene depending on its cellular localization. Whereas the growth inhibitory functions of p21 are associated with its nuclear localization, the anti-apoptotic or oncogenic activities are frequently associated with its cytoplasmic accumulation [12,13]. When p21 is localized to the nucleus it arrests cell growth by inhibiting cyclin dependent kinases [14], and also by inhibiting DNA synthesis through interactions with proliferating cell nuclear antigen, PCNA [15]. In contrast, in the cytoplasm, p21 complexes with and inactivates Apoptosis Signaling Kinase 1 (ASK1) [12,16], resulting in the activation of anti-apoptotic BCL-2 [17]. Our investigations have revealed that in SUM159, p53-null, triple-negative, inflammatory breast cancer cells [18] NUPR1-driven signaling activates PI3K/AKT signaling and causes p21 to be localized to the cytoplasm thereby resulting in hypo-phosphorylated Bcl-xL,increased cell proliferation and subsequent chemoresistance to doxorubicin. 2. Materials and methods 2.1. Cell lines, expression constructs and transfection SUM159 cells and MCF10AT cells were cultured as previously described [19]. Human NUPR1 cDNA was cloned in pcDNA3.1(+)
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.063
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by PCR amplification of the gene from pEF6/V5-His(p8). SUM159 cells, obtained from Asterand Inc., were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per manufacturer’s specifications. Stable transfectants were validated by real-time qRT-PCR and western blotting. Endogenous NUPR1 from MCF10AT cells (obtained from the Karmanos Cancer Center) was silenced using shRNA (Oligoengine, Seattle, WA). NUPR1 knockdown stable cells were selected in 250 ng/ml puromycin. Stable transfectants were derived from cell cultures with less than 10 passages in order to minimize the possible impact of clonal diversification and phenotypic instability in vitro. 2.2. Real-time quantitative PCR of tissue array TissueScan plates (Origene, Rockville, MD) were assessed for the expression of NUPR1 transcripts using the manufacturer’s protocol. The reaction was carried out in a Bio-Rad iCycler iQ5 using the following program: activation step of 50 °C for 2 min, then 42 cycles of 95 °C for 5 min, 95 °C for 15 s, and 60 °C for 1 min. Data was expressed as dCT (difference in Ct between Ct values of NUPR1 and bactin). A 5% level of significance was used to determine significance of results. 2.3. Chemicals and antibodies Antibodies used in this study include p21 (Cell Signaling Technology, Danvers, MA), phospho-Thr145 p21 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Ser146 p21 (Santa Cruz), GAPDH (Cell Signaling), horseradish peroxidase linked beta-actin (Abcam, Cambridge, MA), phospho-Ser473-Akt (Cell Signaling), pan-Akt (Cell Signaling), BCL-xL (Cell Signaling), phospho-Ser62 BCL-xL (abcam), ECL™ sheep anti-mouse IgG HRP linked (GE Healthcare UK, Limited, Amersham, Buckinghamshire, UK), ECL™ donkey anti-rabbit IgG HRP linked (GE Healthcare UK, Limited), donkey anti-goat IgG HRP linked (Santa Cruz), and Alexa FluorÒ 488 Goat anti-rabbit (Invitrogen). Small molecule inhibitors used include Wortmannin to inhibit PI-3 Kinase and, Akt Inhibitor IV (Calbiochem) to inhibit Akt (IC50<1.25 lM). 3. Immunocytochemistry Cells were plated on Thomas Red Label MICRO Cover Glasses (Thomas Scientific, Swedesboro, NJ), and treated as indicated. Cells were fixed and permeabilized with methanol at 20 °C for 5 min, blocked in 10% normal goat serum in PBS, and stained with 1:200 rabbit anti-p21 overnight at 4 °C. Cells were washed in PBS, and treated with 1:200 Alexa FluorÒ 488 Goat anti-rabbit at room temperature for 1 h. The cells were mounted in VECTASHIELDÒ with propidium iodide (Vector Laboratories, Inc, Burlingame, CA), imaged on a Nikon Eclipse TE-2000U microscope (Nikon Instruments Inc, Melville, NY). We estimated 10 fields per image for quantitation. 4. Results 4.1. NUPR1 modulates chemoresistance and cellular localization of p21 We probed an array comprising cDNA derived from normal breast tissue and breast tumor tissue for the expression of NUPR1. The levels of NUPR1 were upregulated (p < 0.05) in breast tumor tissue relative to normal breast tissue (Fig. 1A). In order to understand the role of NUPR1 in modulating the chemoresponsiveness of breast tumor cells, we worked with two systems: SUM159 cells were genetically engineered to constitutively express NUPR1 and the endogenous NUPR1 in MCF10AT cells was abrogated by RNA
interference. We chose to overexpress NUPR1 in SUM159 cells in order to model the ability of NUPR1 to aid in disease progression by increasing chemoresistance in this already pathologic cell line. On the other hand, we chose to abrogate the expression of NUPR1 in MCF10AT cells to provide evidence for NUPR1’s physiologic ability to protect breast epithelial cells from stress, including chemotherapeutic induced stress. Stable expression of NUPR1 in SUM159 cells resulted in upregulation of p21 to a small extent (Fig. 1B and Supplementary Fig. 1A). Interestingly, p21 in the NUPR1-expressing cells was localized to the cytoplasmic compartment (Fig. 1C and Supplementary Fig. 1B). The upregulation of NUPR1 achieved in these cells is 6-fold, comparable to the physiological upregulation in NUPR1 levels that occur when cells are treated with stressors such as TNF, ceramide, staurosporine or LPS [20,21]. In agreement with our previous reports, NUPR1 expression allowed the cells to resist the cytotoxic effects of doxorubicin (Fig. 1D; Supplementary Fig. 1C). In contrast, MCF10AT cells that were engineered for stable silencing of endogenous NUPR1 (Fig. 1E) showed enhanced cytotoxicity in response to doxorubicin (Fig. 1F) and taxol (Supplementary Fig. 1D). While the vector control cells showed diffuse distribution of p21, in the NUPR1-silenced cells, p21 was localized predominantly in the nucleus (Fig. 1G and Supplementary Fig. 2).Thus, modulating NUPR1 expression in cells altered the sub-cellular localization of p21. 4.2. PI-3-K/Akt signaling mediates the cytoplasmic localization of p21 in NUPR1-expressing cells While nuclear p21 executes a tumor suppressive function, cytoplasmic p21 functions in an anti-apoptotic role and enhances cell survival. The mechanisms that regulate the cytoplasmic localization of p21 have been mainly attributed to the phosphorylation of consensus sites Thr145 and/or Ser146 within the nuclear localization signal (NLS) of p21 [22–24]. The Protein Kinase B/Akt pathway is attributed to cause phosphorylation of p21 at its NLS resulting in cytoplasmic localization of p21 [22,25,26]. Besides Akt, IjB kinase b (IKKb) has also been reported to bring about cytoplasmic localization of p21 [27]. IKKb activity also causes phosphorylation of Akt [27] and subsequent cytoplasmic p21 accumulation. IKKb is one of the two catalytic subunits of the IKK complex. Upon activation by cytokines, the IKK complex, mainly IKKb, signals to activate the transcription factor, NF-jB. IKKb activity also causes phosphorylation of Akt [27] and subsequent cytoplasmic p21 accumulation. To determine the mechanism(s) that lead to NUPR1-mediated altered localization of p21, we assessed the impact of an Akt inhibitor (Akt Inhibitor IV), a PI-3 -kinase inhibitor (wortmannin) and an IKK inhibitor (BMS-345541) on NUPR1-expressing cells and control cells and performed immunofluorescent microscopy to visualize the impact on sub-cellular localization of p21. NUPR1 overexpression corresponded to nuclear exclusion of p21 with a resultant cytoplasmic localization of p21. In contrast, control cells displayed predominantly nuclear p21 staining. While Akt Inhibitor IV and Wortmannin reversed the NUPR1-mediated cytoplasmic-localization of p21, the IKK inhibitor was only partially effective at restoring the nuclear localization of p21 (Fig. 2). 4.3. NUPR1 expression results in Akt-mediated phosphorylation of p21 In order to determine if specific post-translational modifications of p21 result from the expression of signaling initiated by NUPR1, we assessed the NUPR1-expressing and vector control cells for their status of Akt and p21. As seen in Fig. 3A, while NUPR1 expression results in an overall increase in the levels of Akt, there is also a distinct increase in the levels of activated p-Ser-473-Akt
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Fig. 1. NUPR1 modulates p21 localization and chemoresponsiveness of breast cancer cells. (A) NUPR1 transcript levels are upregulated in breast tumor tissues relative to normal breast tissue (⁄p = 0.03). (B) Stable transfectants (clones 15 and 37) of SUM159 cells expressing NUPR1 were generated. Cell lysates were resolved by SDS-PAGE and immunoblotted. NUPR1-expresing cells show minimal changes in the levels of p21. b-Actin serves as a loading control. UT: Untransfected cells; EV: empty vector transfectants. (C) NUPR1 expression in SUM159 cells causes p21 to be predominantly localized to the cytoplasm. The cells were stained for p21 (FITC) and DAPI and assessed microscopically for p21 localization by immunocytochemistry. (D) NUPR1 expression makes SUM159 cells resistant (⁄p < 0.05) to doxorubicin. Cells were treated with doxorubicin (0.5 lM) for 72 h and viability was assessed by MTS assay. (E) Endogenous NUPR1 in MCF10AT cells was silenced by RNA interference. 187-3, -6, -9 denote stable clones that are silenced for NUPR1 using shRNA187. EV denotes empty vector transfectants and SC denotes cells transfected with a non-targeting control shRNA. Loss of NUPR1 is accompanied by a notable change in the levels of p21. (F). Abrogating endogenous NUPR1 increased the sensitivity of the cells to doxorubicin (0.5 lM for 72 h) (⁄p < 0.05). (G) Relative to the MCF10AT cells transfected with empty vector (EV) and non-targeting control shRNA (SC), the NUPR1-silenced cells (shNUPR1) show nuclear localization of p21 (FITC) when assessed by immunocytochemistry. DAPI serves to label the nuclei.
that is concomitant with an increase in P-Thr-145-p21. NUPR1overexpressing cells also showed an overall increase in BCL-xL levels. However, the deactivated phosphorylated 62-serine form of BCL-xL was barely detectable in NUPR1-expressing cells in comparison to vector control cells [28]. We did not detect any change in the P-Ser-146-p21 levels. Phosphorylation of p21 at Ser 146 and/or Thr 145 is associated with cytoplasmic localization of p21 and lead to activated Bcl-xL. In order to assess a role for activated PI-3-K/Akt in mediating phosphorylation of p21 at the Thr145 and/ or Ser146 residues we treated the vector control and NUPR1expressing cells with inhibitors of Akt (Akt Inhibitor IV) and PI-3kinase (wortmannin). We also assessed a role for IKK (NF-jB signaling) using BMS-34551. Treatment with an Akt inhibitor resulted in reduced levels of activated p-Ser-473-Akt with concomitantly decreased levels of p-Thr-145-p21. This was accompanied by an increase in p-Ser-62-Bcl-xL, indicating an increase in the cellular pools of inactive Bcl-XL (Fig. 3 A& B). Notably, while inhibition of NF-jB signaling had no impact on the total cellular pool of p21, inhibition of PI-3-kinase and Akt caused a drastic decrease in the levels of total p21. While not completely resolved, there are a couple of reports documenting a role for Akt in stabilizing p21 [29,30]; and the withdrawal of or interference in Akt signaling destabilizes p21 by targeting it for proteasomal degradation [30]. Inhibition of IKK with BMS-345541 did not result in any notable change in the levels of phosphorylated Akt or p-Thr-145-p21. There was a minimal change in the levels of p-Ser-62-Bcl-xL. Inhibition of PI-3-kinase using wortmannin greatly impacted the activation of Akt as
seen by a complete absence of p-Ser-473-Akt in the treated NUPR1-expressing cells. There was a simultaneous profound reduction in the p-Thr-145-p21 levels. Surprisingly, the pools of p-Ser-62-Bcl-xL remained low in the NUPR1-expressing cells despite wortmannin treatment. The 146-Serine residue of p21 was not affected by any variables imposed in this study. Taken together, these results support our claims that NUPR1 mediates cytoplasmic localization of p21 by phosphorylating its nuclear localization signal in a PI-3 Kinase/Akt dependent manner. In addition, this correlated with subsequent activation of anti-apoptotic Bcl-xL. 4.4. Inhibition of Akt and PI-3K sensitizes NUPR1-expressing cells to doxorubicin In order to understand the determinative role of NUPR1-induced PI-3-K/Akt-mediated phosphorylation and cytoplasmic localization of p21 with the chemoresistance properties of the cells, we assessed the response of the cells to doxorubicin when co-treated with inhibitors of Akt, PI-3-K and IKK. As seen in Fig. 4A and B, while inhibition of Akt and PI-3-kinase restored the sensitivity of the NUPR1-expressing cells to doxorubicin, the NUPR1-expressing cells remained resistant to doxorubicin even in presence of the IKK inhibitor, indicating that the chemoresistance imparted by NUPR1 is independent of the NF-jB pathway (Fig. 4C). Pre-treatment with any of the inhibitors did not impact the response to doxorubicin (data not shown).
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Fig. 2. Blocking PI3K/Akt signaling reverses NUPR1-mediated p21 cytoplasmic localization in SUM159 cells. SUM159 cells were plated on coverslips and treated for 24 h with the indicated inhibitor (Akt Inhibitor IV l lM, or Wortmannin l lM or BMS-345541, l lM). Cells were fixed and stained as indicated in Methods and Materials. Images acquired at 40 magnification. NUPR1 overexpressing cells displayed predominantly cytoplasmic localization of p21, which was reversed by both Akt Inhibitor IV and Wortmannin treatment. Representative images are shown. The ratio of nuclear:cytoplasmic signal of p21 was determined from these images. Mean intensity of p21 staining was determined for each cell in both the cell nucleus and the whole cell. Cytoplasmic intensity was determined indirectly by subtracting the summed nuclear intensity from the summed cellular intensity and controlling for cellular volume. A ratio was then taken for each cell comparing the mean intensity of the cell nucleus to the mean intensity of the cytoplasm. p21 localization becomes similar after treatment with Wortmannin and Akt Inhibitor IV.
Fig. 3. NUPR1 leads to phosphorylation of p21 via intermediates PI-3 Kinase and Akt and is correlated with increased levels of hypophosphorylated BCL-xL. (A) NUPR1 overexpressing SUM159 cells displayed increased total Akt and the active phosphorylated form of Akt (phospho-473-Serine Akt). These cells also showed increased phosphorylation of p21 at the 145-Threonine residue but not at the 146-Serine residue. Treatment for 2 h with Akt and PI-3 Kinase inhibitors (Akt Inhibitor IV and Wortmannin, respectively) reversed the phosphorylation status of both Akt and p21. Inhibition of IKK using BMS-345541 did not impact the phosphorylation of p21. NUPR1 overexpressing SUM159 cells also demonstrated increased levels of BCL-xL but decreased phosphorylated 62-Serine signal. Cells treated with Akt Inhibitor or Wortmannin for 24 h showed an increase in the levels of phosphorylated BCL-xL. (B) Densiometric analysis of p21 phosphorylation status reveals decrease in p21 phosphorylation of the 145Threonine residue after treating NUPR1 overexpressing cells with Akt Inhibitor IV and Wortmannin, but not IKK inhibitor. The levels of Bcl-xL phosphorylation (indicating inactive Bcl-xL) is increased when the NUPR1-expressing cells are treated with Akt inhibitor relative to untreated cells. Wortmannin or BMS-345541 treatment does not impact Bcl-xL phosphorylation.
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Fig. 4. NUPR1 mediates chemoresponsiveness via PI-3-K/Akt. SUM159 cells transfected with empty vector or NUPR1 were treated with doxorubicin (0.5 lM) and/or Akt inhibitor IV (1 lM) or wortmannin (1 lM) or IKK inhibitor (1 lM) and cell viability was assessed 24 h later by MTS assay. The NUPR1 cells demonstrated increased proliferation in culture (untreated). While the inhibitors alone did not impact cell viability, when co-treated with (A) Akt inhibitor or (B) wortmannin, the NUPR1-expressing cells regained sensitivity to doxorubicin. (C) Inhibition of IKK did not impact cellular response to doxorubicin (even when higher concentrations of the IKK inhibitor were used; data not shown).
5. Discussion NUPR1 was first identified as a protein physiologically involved in the cellular stress response, promoting regeneration and cellular growth [31]. NUPR1 functions in the nucleus where it serves as a co-factor for transcription factors such as p53 [9] and estrogen receptor-b [32]. Since NUPR1 is a stress molecule that might be upregulated in actual in vivo conditions, where the tumor cells undergo selection pressure, stress from the surrounding cells and from the host’s defense mechanisms may play critical roles in influencing the activity of NUPR1. The potential of NUPR1 in making cancer cells resistant to the effects of conventional chemotherapeutic agents is suggested by two independent studies examining the effects of NUPR1 expression in pancreatic [33] and breast cancer [9]. Gemcitabine, the drug of choice for pancreatic cancer, was found to target NUPR1. Gemcitabine resistant pancreatic cancer cells devoid of NUPR1 (NUPR1 / ) were more prone to apoptosis than their wild type counterparts, and gemcitabine-induced apoptosis mainly resulted from the inhibition of the constitutive antiapoptotic activity of NUPR1 [3]. Alternatively, named as cyclin-dependent kinase inhibitor-IA (CDKN1A), CDK-interacting protein 1 (CIP1) and wild-type p53activated fragment 1 (WAF1), p21 is a protein possessing duality in the regulation of cancer behavior. The nuclear form of p21 mainly executes its tumor suppressing function by binding to CDK complexes, inhibiting the activity of cyclin-dependent kinases, such as, Cdk2, Cdk3, Cdk4 and Cdk6, leading to cell growth arrest [14]. In contrast, the cytoplasmic form of p21 complexes with and inactivates pro-apoptotic proteins such as caspase-3 [12] and ASK-1 [13,34]. This oncogenic function of p21 has also been validated by clinical evidence. Higher expression of cytoplasmic p21 has been verified as a factor of poor prognosis in human primary breast carcinomas [23,35]. Moreover, resistance to chemotherapeutic drugs may result from accumulation of cytoplasmic p21, as paclitaxel could increase cytoplasmic p21, and reverse the cell cycle inhibitory effect of p21 [25,26]. Thus, cellular localization plays a critical role to determine the function of p21, i.e. nuclear p21, as a cell cycle inhibitor, represses cell growth; cytoplasmic p21, as an anti-apoptotic factor, enhances cell survival. Phosphorylation of p21 at Ser 146 and/or Thr 145 is associated with cytoplasmic localization of p21 [22]. Activated Akt phosphorylates p21 at Ser 146 and stabilizes p21; phosphorylation of p21 at the Thr 145 position makes p21 a positive modulator of cell survival and proliferation. Specifically, phosphorylated p21 does not inhibit cyclin E-CDK2 but instead, promotes the binding and assembly and activation of the cyclin D1-CDK4 complex, which is a critical mediator of G1-S progression, acting epistatic to and inhibiting p27. Phosphorylation of
p21 at Thr145 releases PCNA from p21 binding and resultant inhibition. This release allows for elevated p21 levels to exist in cells without inhibiting the cell’s ability to progress through S phase [30,36]. As such, phosphorylation of p21 at these two critical residues not only alters its sub-cellular localization, but also interferes in the growth-restrictive properties of p21 so profoundly, making it a pro-survival molecule. We have previously shown that p53 and p300 are required for NUPR1 mediated up-regulation of p21 [9]. Mutations in p53 are seen in about 30% of breast cancers. Triple-negative basal-like breast cancers account for 15% of breast cancers and have an increased frequency of p53 mutations. Mutant p53 status is a strongly unfavorable prognostic indicator in these patients for overall survival and response to anthracycline-containing therapy. Inflammatory breast cancer (T4d in the TNM classification) has a particularly aggressive behavior and poor prognosis, and mutations in p53 are more frequent (50%) in inflammatory than in non-inflammatory breast cancer (20–30%) [37]. The SUM159PT cell line, derived from inflammatory breast cancer contains a p53 loss of function missense mutation [18], and is triple-negative for ER, PR, and HER2/Neu [38]. Thus, it presents a unique system to study the role of NUPR1 in chemoresistance. In these cells NUPR1 expression did not result in an appreciable increase in p21, but rather resulted in altered localization of p21. Thus, the regulation of sub-cellular localization of p21 mediated by Akt is, from the perspective of NUPR1-induced chemoresistance, a novel finding. Akt has been well studied for its role in inhibiting apoptosis and promoting cell survival. The PI-3K-Akt pathway has been linked to resistance to a variety of chemotherapeutics, such as gemcitabine, irinotecan, etoposide, and Taxol [39–43]. Hyperphosphorylation of Akt has also been linked to poor prognosis in a variety of cancers [44]. Our results show that p21 that is phosphorylated by Akt is unable to carry out its tumor suppressive functions, and instead causes activation of BCL-xL available in the cellular pool to complex with and de-activate pro-apoptotic Bax. NUPR1 overexpression increases cellular proliferation, while removing necessary cell cycle checkpoints [9], and our findings elucidate a NUPR1PI-3-K/Akt-phospho-p21 axis that enhances chemoresistance in breast cancer cells. Various PI-3-K/Akt inhibitors are in clinical trials. It is possible that the use of such inhibitors may help restore chemosensitivity in patients whose tumors show upregulated NUPR1 expression, thereby expanding the scope of the treatment. 6. Grant support We gratefully acknowledge funding from the Norwegian Radium Research Foundation. This study was performed at the University of South Alabama Mitchell Cancer Institute.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.07. 063. References [1] Hortobagyi, G.N., Heim, W., Hutchins, L., Rivera, E., Mason, B., Booser, D.J. and Kirshner, J. (2010) A phase 2 study of a fixed combination of uracil and ftorafur (UFT) and leucovorin given orally in a 3-times-daily regimen to treat patients with recurrent metastatic breast cancer. Cancer 116, 1440–1445. [2] Castiglione-Gertsch, M., The International Breast Cancer Study Group, et al. (1997) Retreating recurrent breast cancer with the same CMF-containing regimen used as adjuvant therapy. Eur. J. Cancer 33, 2321–2325. [3] Giroux, V., Malicet, C., Barthet, M., Gironella, M., Archange, C., Dagorn, J.C., Vasseur, S. and Iovanna, J.L. (2006) p8 is a new target of gemcitabine in pancreatic cancer cells. Clin. Cancer Res. 12, 235–241. [4] Su, S.B. et al. (2001) Expression of p8 in human pancreatic cancer. Clin. Cancer Res. 7, 309–313. [5] Ree, A.H. et al. (2002) Clinical and cell line specific expression profiles of a human gene identified in experimental central nervous system metastases. Anticancer Res. 22, 1949–1957. [6] Ito, Y. et al. (2003) Expression and cellular localization of p8 protein in thyroid neoplasms. Cancer Lett. 201, 237–244. [7] Mohammad, H.P., Seachrist, D.D., Quirk, C.C. and Nilson, J.H. (2004) Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing hormone. Mol. Endocrinol. 18, 2583–2593. [8] Kong, D.K., Georgescu, S.P., Cano, C., Aronovitz, M.J., Iovanna, J.L., Patten, R.D., Kyriakis, J.M. and Goruppi, S. (2010) Deficiency of the transcriptional regulator p8 results in increased autophagy and apoptosis, and causes impaired heart function. Mol. Biol. Cell 21, 1335–1349. [9] Clark, D.W., Mitra, A., Fillmore, R.A., Jiang, W.G., Samant, R.S., Fodstad, O. and Shevde, L.A. (2008) NUPR1 interacts with p53, transcriptionally regulates p21 and rescues breast epithelial cells from doxorubicin-induced genotoxic stress. Curr. Cancer Drug Targets 8, 421–430. [10] Ree, A.H. et al. (1999) Expression of a novel factor in human breast cancer cells with metastatic potential. Cancer Res. 59, 4675–4680. [11] Sandi, M.J., Hamidi, T., Malicet, C., Cano, C., Loncle, C., Pierres, A., Dagorn, J.C. and Iovanna, J.L. (2011) p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis. J. Cell Physiol. 226, 3442– 3451. [12] Asada, M., Yamada, T., Ichijo, H., Delia, D., Miyazono, K., Fukumuro, K. and Mizutani, 5. (1999) Apoptosis inhibitory activity of cytoplasmic p21(Cip1/ WAF1) in monocyticdifferentiation. EMBO J. 18, 1223–1234. [13] Blagosklonny, M.V. (2002) Are p27 and p21 cytoplasmic oncoproteins? Cell Cycle 1, 391–393. [14] Harper, J.W. et al. (1995) Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell 6, 387–400. [15] Flores-Rozas, H., Kelman, Z., Dean, F.B., Pan, Z.Q., Harper, J.W., Elledge, S.J., O’Donnell, M. and Hurwitz, J. (1994) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc. Natl. Acad. Sci. USA 91, 8655– 8659. [16] Zhan, J., Easton, J.B., Huang, S., Mishra, A., Xiao, L., Lacy, E.R., Kriwacki, R.W. and Houghton, P.J. (2007) Negative regulation of ASK1 by p21Cip1 involves a small domain that includes Serine 98 that is phosphorylated by ASK1 in vivo. Mol. Cell. Biol. 27, 3530–3541. [17] Yamamoto, K., Ichijo, H. and Korsmeyer, S.J. (1999) BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol. Cell. Biol. 19, 8469–8478. [18] Wasielewski, M., Elstrodt, F., Klijn, J.G., Berns, E.M. and Schutte, M. (2006) Thirteen new p53 gene mutants identified among 41 human breast cancer cell lines. Breast Cancer Res. Treat. 99, 97–101. [19] Morrow, K.A., Das, S., Metge, B.J., Ye, K., Mulekar, M.S., Tucker, J.A., Samant, R.S. and Shevde, L.A. (2011) Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation. J. Biol. Chem. 286, 40376– 40385. [20] Vasseur, S., Vidal Mallo, G., Fiedler, F., Bodeker, H., Canepa, E., Moreno, S. and Iovanna, J.L. (1999) Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur. J. Biochem. 259, 670–675. [21] Jiang, Y.F., Vaccaro, M.I., Fiedler, F., Calvo, E.L. and Iovanna, J.L. (1999) Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro. Biochem. Biophys. Res. Commun. 260, 686–690.
[22] Zhou, B.P., Liao, Y., Xia, W., Spohn, B., Lee, M.H. and Hung, M.C. (2001) Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER- 2/neu-overexpressing cells. Nat. Cell. Biol. 3, 245–252. [23] Xia, W., Chen, J.S., Zhou, X., Sun, P.R., Lee, D.F., Liao, Y., Zhou, B.P. and Hung, M.C. (2004) Phosphorylation/cytoplasmic localization of p21Cip1/WAF1 is associated with HER2/neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients. Clin. Cancer Res. 10, 3815–3824. [24] Yuste, L., Montero, J.C., Esparis-Ogando, A. and Pandiella, A. (2005) Activation of ErbB2 by overexpression or by transmembrane neuregulin results in differential signaling and sensitivity to herceptin. Cancer Res. 65, 6801–6810. [25] Perez-Tenorio, G., Berglund, F., Esguerra Merca, A., Nordenskjold, B., Rutqvist, L.E., Skoog, L. and Stal, O. (2006) Cytoplasmic p21WAF1/CIP1 correlates with Akt activation and poor response to tamoxifen in breast cancer. Int. J. Oncol. 28, 1031–1042. [26] Heliez, C., Baricault, L., Barboule, N. and Valette, A. (2003) Paclitaxel increases p21 synthesis and accumulation of its AKT-phosphorylated form in the cytoplasm of cancer cells. Oncogene 22, 3260–3268. [27] Ping, B. et al. (2006) Cytoplasmic expression of p21CIP1/WAF1 is correlated with IKKbeta overexpression in human breast cancers. Int. J. Oncol. 29, 1103– 1110. [28] Upreti, M., Galitovskaya, E.N., Chu, R., Tackett, A.J., Terrano, D.T., Granell, S. and Chambers, T.C. (2008) Identification of the major phosphorylation site in BclxL induced by microtubule inhibitors and analysis of its functional significance. J. Biol. Chem. 283, 35517–35525. [29] Park, J.K., Cho, C.H., Ramachandran, S., Shin, S.J., Kwon, S.H., Kwon, S.Y. and Cha, S.D. (2006) Augmentation of sodium butyrate-induced apoptosis by phosphatidylinositol 3-kinase inhibition in the human cervical cancer cellline. Cancer Res. Treat. 38, 112–117. [30] Li, Y., Dowbenko, D. and Lasky, L.A. (2002) AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277, 11352–11361. [31] Mallo, G.V., Fiedler, F., Calvo, E.L., Ortiz, E.M., Vasseur, S., Keim, V., Morisset, J. and Iovanna, J.L. (1997) Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth. J. Biol. Chem. 272, 32360–32369. [32] Jiang, W.G., Davies, G. and Fodstad, O. (2005) Com-1/P8 in oestrogen regulated growth of breast cancer cells, the ER-beta connection. Biochem. Biophys. Res. Commun. 330, 253–262. [33] Vasseur, S. et al. (2004) p8 improves pancreatic response to acute pancreatitis by enhancing the expression of the anti-inflammatory protein pancreatitisassociated protein I. J. Biol. Chem. 279, 7199–7207. [34] Suzuki, A., Tsutomi, Y., Akahane, K., Araki, T. and Miura, M. (1998) Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene 17, 931–939. [35] Winters, Z.E., Hunt, N.C., Bradburn, M.J., Royds, J.A., Turley, H., Harris, A.L. and Norbury, C.J. (2001) Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/ CIP1) in breast cancer association with prognosis. Eur. J. Cancer 37, 2405– 2412. [36] Malumbres, M. and Barbacid, M. (2006) Is Cyclin D1-CDK4 kinase a bona fide cancer target? Cancer Cell 9, 2–4. [37] Varna, M., Bousquet, G., Plassa, L.F., Bertheau, P. and Janin, A. (2011) TP53 status and response to treatment in breast cancers. J. Biomed. Biotechnol., 284584. [38] Hollestelle, A. et al. (2010) Distinct gene mutation profiles among luminaltype and basal-type breast cancer cell lines. Breast Cancer Res. Treat. 121, 53– 64. [39] Campbell, R.A., Bhat-Nakshatri, P., Patel, N.M., Constantinidou, D., Ali, S. and Nakshatri, H. (2001) Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J. Biol. Chem. 276, 9817–9824. [40] Perez-Tenorio, G. and Stal, O. (2002) Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br. J. Cancer 86, 540–545. [41] Shah, A. et al. (2005) Phospho-akt expression is associated with a favorable outcome in non-small cell lung cancer. Clin. Cancer Res. 11, 2930–2936. [42] Bleau, A.M., Hambardzumyan, D., Ozawa, T., Fomchenko, E.I., Huse, J.T., Brennan, C.W. and Holland, E.C. (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 4, 226–235. [43] Sinnberg, T. et al. (2009) Inhibition of PI3K-AKT-mTOR signaling sensitizes melanoma cells to cisplatin and temozolomide. J. Invest. Dermatol. 129, 1500– 1515. [44] Cheng, J.Q., Lindsley, C.W., Cheng, G.Z., Yang, H. and Nicosia, S.V. (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24, 7482–7492.
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Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance NUPR1 and p21 in chemoresistance Andrew J. Vincent a, Suping Ren a, Lillianne G. Harris a, Daniel J. Devine a, Rajeev S. Samant b,c, Oystein Fodstad d,e, Lalita A. Shevde b,c,⇑ a
Mitchell Cancer Institute, University of South Alabama, Mobile, AL, United States Department of Pathology, University of Alabama at Birmingham, United States Comprehensive Cancer Center, University of Alabama at Birmingham, United States d Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway e Institute for Clinical Medicine, Faculty of Medicine, University in Oslo, Oslo, Norway b c
a r t i c l e
i n f o
Article history: Received 29 March 2012 Revised 9 July 2012 Accepted 22 July 2012 Available online 31 July 2012 Edited by Varda Rotter Keywords: NUPR1 P21 Chemoresistance Akt IKK-b
a b s t r a c t The expression of Nuclear Protein 1 (NUPR1) is associated with chemoresistance in multiple malignancies. We previously reported that NUPR1 functions as a transcriptional cofactor for the p300–p53 complex and transcriptionally regulates p21 expression. In the present study we investigated the activity of NUPR1 in p53-deficient, triple-negative, inflammatory SUM159 breast cancer cells. Our studies reveal that NUPR1 confers growth benefit and chemoresistance by causing Akt-mediated phosphorylation and subsequent cytoplasmic re-localization of p21 and activation of the anti-apoptotic Bcl-xL protein. Our findings elucidate a NUPR1-PI-3-K/Akt-phospho-p21 axis that functions in p53-negative, inflammatory breast cancer cells to enhance chemoresistance in breast cancer. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Much progress has been made in the treatment of breast cancer, but in metastatic disease chemoresistance remains a barrier to long-term survival. A number of clinical trials have demonstrated that complete response occurs in only one-third of patients with recurrent breast cancer [1,2]. Hence, there is an urgent need to identify molecular mechanisms that regulate chemoresistance. Nuclear Protein 1 (NUPR1, also known as com1 and p8) is a small molecule whose levels are upregulated in response to cellular stress and also in multiple malignancies [3–7] and are inversely correlated with apoptosis and autophagy [8]. We and others have shown that NUPR1 confers resistance to multiple drugs, including gemcitabine, paclitaxel and doxorubicin [9]. NUPR1 plays a role in tumor progression and distant metastasis of breast cancer [10] and is shown to regulate many steps in metastasis, including migration, invasion, and adhesion [11]. We have reported that NUPR1 mediates doxorubicin resistance in a p53-dependent manner by transcriptionally upregulating p21 [9]. p21 is a well-known negative regulator of cell cycle progression. ⇑ Corresponding author. Address: Department of Pathology, University of Alabama at Birmingham, 1824 6th Avenue South, Birmingham, AL 35233, United States. Fax: +1 205 975 6615. E-mail address: [email protected] (L.A. Shevde).
Recent studies show that p21 can act both as a tumor suppressor gene and an oncogene depending on its cellular localization. Whereas the growth inhibitory functions of p21 are associated with its nuclear localization, the anti-apoptotic or oncogenic activities are frequently associated with its cytoplasmic accumulation [12,13]. When p21 is localized to the nucleus it arrests cell growth by inhibiting cyclin dependent kinases [14], and also by inhibiting DNA synthesis through interactions with proliferating cell nuclear antigen, PCNA [15]. In contrast, in the cytoplasm, p21 complexes with and inactivates Apoptosis Signaling Kinase 1 (ASK1) [12,16], resulting in the activation of anti-apoptotic BCL-2 [17]. Our investigations have revealed that in SUM159, p53-null, triple-negative, inflammatory breast cancer cells [18] NUPR1-driven signaling activates PI3K/AKT signaling and causes p21 to be localized to the cytoplasm thereby resulting in hypo-phosphorylated Bcl-xL,increased cell proliferation and subsequent chemoresistance to doxorubicin. 2. Materials and methods 2.1. Cell lines, expression constructs and transfection SUM159 cells and MCF10AT cells were cultured as previously described [19]. Human NUPR1 cDNA was cloned in pcDNA3.1(+)
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.063
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by PCR amplification of the gene from pEF6/V5-His(p8). SUM159 cells, obtained from Asterand Inc., were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per manufacturer’s specifications. Stable transfectants were validated by real-time qRT-PCR and western blotting. Endogenous NUPR1 from MCF10AT cells (obtained from the Karmanos Cancer Center) was silenced using shRNA (Oligoengine, Seattle, WA). NUPR1 knockdown stable cells were selected in 250 ng/ml puromycin. Stable transfectants were derived from cell cultures with less than 10 passages in order to minimize the possible impact of clonal diversification and phenotypic instability in vitro. 2.2. Real-time quantitative PCR of tissue array TissueScan plates (Origene, Rockville, MD) were assessed for the expression of NUPR1 transcripts using the manufacturer’s protocol. The reaction was carried out in a Bio-Rad iCycler iQ5 using the following program: activation step of 50 °C for 2 min, then 42 cycles of 95 °C for 5 min, 95 °C for 15 s, and 60 °C for 1 min. Data was expressed as dCT (difference in Ct between Ct values of NUPR1 and bactin). A 5% level of significance was used to determine significance of results. 2.3. Chemicals and antibodies Antibodies used in this study include p21 (Cell Signaling Technology, Danvers, MA), phospho-Thr145 p21 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Ser146 p21 (Santa Cruz), GAPDH (Cell Signaling), horseradish peroxidase linked beta-actin (Abcam, Cambridge, MA), phospho-Ser473-Akt (Cell Signaling), pan-Akt (Cell Signaling), BCL-xL (Cell Signaling), phospho-Ser62 BCL-xL (abcam), ECL™ sheep anti-mouse IgG HRP linked (GE Healthcare UK, Limited, Amersham, Buckinghamshire, UK), ECL™ donkey anti-rabbit IgG HRP linked (GE Healthcare UK, Limited), donkey anti-goat IgG HRP linked (Santa Cruz), and Alexa FluorÒ 488 Goat anti-rabbit (Invitrogen). Small molecule inhibitors used include Wortmannin to inhibit PI-3 Kinase and, Akt Inhibitor IV (Calbiochem) to inhibit Akt (IC50<1.25 lM). 3. Immunocytochemistry Cells were plated on Thomas Red Label MICRO Cover Glasses (Thomas Scientific, Swedesboro, NJ), and treated as indicated. Cells were fixed and permeabilized with methanol at 20 °C for 5 min, blocked in 10% normal goat serum in PBS, and stained with 1:200 rabbit anti-p21 overnight at 4 °C. Cells were washed in PBS, and treated with 1:200 Alexa FluorÒ 488 Goat anti-rabbit at room temperature for 1 h. The cells were mounted in VECTASHIELDÒ with propidium iodide (Vector Laboratories, Inc, Burlingame, CA), imaged on a Nikon Eclipse TE-2000U microscope (Nikon Instruments Inc, Melville, NY). We estimated 10 fields per image for quantitation. 4. Results 4.1. NUPR1 modulates chemoresistance and cellular localization of p21 We probed an array comprising cDNA derived from normal breast tissue and breast tumor tissue for the expression of NUPR1. The levels of NUPR1 were upregulated (p < 0.05) in breast tumor tissue relative to normal breast tissue (Fig. 1A). In order to understand the role of NUPR1 in modulating the chemoresponsiveness of breast tumor cells, we worked with two systems: SUM159 cells were genetically engineered to constitutively express NUPR1 and the endogenous NUPR1 in MCF10AT cells was abrogated by RNA
interference. We chose to overexpress NUPR1 in SUM159 cells in order to model the ability of NUPR1 to aid in disease progression by increasing chemoresistance in this already pathologic cell line. On the other hand, we chose to abrogate the expression of NUPR1 in MCF10AT cells to provide evidence for NUPR1’s physiologic ability to protect breast epithelial cells from stress, including chemotherapeutic induced stress. Stable expression of NUPR1 in SUM159 cells resulted in upregulation of p21 to a small extent (Fig. 1B and Supplementary Fig. 1A). Interestingly, p21 in the NUPR1-expressing cells was localized to the cytoplasmic compartment (Fig. 1C and Supplementary Fig. 1B). The upregulation of NUPR1 achieved in these cells is 6-fold, comparable to the physiological upregulation in NUPR1 levels that occur when cells are treated with stressors such as TNF, ceramide, staurosporine or LPS [20,21]. In agreement with our previous reports, NUPR1 expression allowed the cells to resist the cytotoxic effects of doxorubicin (Fig. 1D; Supplementary Fig. 1C). In contrast, MCF10AT cells that were engineered for stable silencing of endogenous NUPR1 (Fig. 1E) showed enhanced cytotoxicity in response to doxorubicin (Fig. 1F) and taxol (Supplementary Fig. 1D). While the vector control cells showed diffuse distribution of p21, in the NUPR1-silenced cells, p21 was localized predominantly in the nucleus (Fig. 1G and Supplementary Fig. 2).Thus, modulating NUPR1 expression in cells altered the sub-cellular localization of p21. 4.2. PI-3-K/Akt signaling mediates the cytoplasmic localization of p21 in NUPR1-expressing cells While nuclear p21 executes a tumor suppressive function, cytoplasmic p21 functions in an anti-apoptotic role and enhances cell survival. The mechanisms that regulate the cytoplasmic localization of p21 have been mainly attributed to the phosphorylation of consensus sites Thr145 and/or Ser146 within the nuclear localization signal (NLS) of p21 [22–24]. The Protein Kinase B/Akt pathway is attributed to cause phosphorylation of p21 at its NLS resulting in cytoplasmic localization of p21 [22,25,26]. Besides Akt, IjB kinase b (IKKb) has also been reported to bring about cytoplasmic localization of p21 [27]. IKKb activity also causes phosphorylation of Akt [27] and subsequent cytoplasmic p21 accumulation. IKKb is one of the two catalytic subunits of the IKK complex. Upon activation by cytokines, the IKK complex, mainly IKKb, signals to activate the transcription factor, NF-jB. IKKb activity also causes phosphorylation of Akt [27] and subsequent cytoplasmic p21 accumulation. To determine the mechanism(s) that lead to NUPR1-mediated altered localization of p21, we assessed the impact of an Akt inhibitor (Akt Inhibitor IV), a PI-3 -kinase inhibitor (wortmannin) and an IKK inhibitor (BMS-345541) on NUPR1-expressing cells and control cells and performed immunofluorescent microscopy to visualize the impact on sub-cellular localization of p21. NUPR1 overexpression corresponded to nuclear exclusion of p21 with a resultant cytoplasmic localization of p21. In contrast, control cells displayed predominantly nuclear p21 staining. While Akt Inhibitor IV and Wortmannin reversed the NUPR1-mediated cytoplasmic-localization of p21, the IKK inhibitor was only partially effective at restoring the nuclear localization of p21 (Fig. 2). 4.3. NUPR1 expression results in Akt-mediated phosphorylation of p21 In order to determine if specific post-translational modifications of p21 result from the expression of signaling initiated by NUPR1, we assessed the NUPR1-expressing and vector control cells for their status of Akt and p21. As seen in Fig. 3A, while NUPR1 expression results in an overall increase in the levels of Akt, there is also a distinct increase in the levels of activated p-Ser-473-Akt
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Fig. 1. NUPR1 modulates p21 localization and chemoresponsiveness of breast cancer cells. (A) NUPR1 transcript levels are upregulated in breast tumor tissues relative to normal breast tissue (⁄p = 0.03). (B) Stable transfectants (clones 15 and 37) of SUM159 cells expressing NUPR1 were generated. Cell lysates were resolved by SDS-PAGE and immunoblotted. NUPR1-expresing cells show minimal changes in the levels of p21. b-Actin serves as a loading control. UT: Untransfected cells; EV: empty vector transfectants. (C) NUPR1 expression in SUM159 cells causes p21 to be predominantly localized to the cytoplasm. The cells were stained for p21 (FITC) and DAPI and assessed microscopically for p21 localization by immunocytochemistry. (D) NUPR1 expression makes SUM159 cells resistant (⁄p < 0.05) to doxorubicin. Cells were treated with doxorubicin (0.5 lM) for 72 h and viability was assessed by MTS assay. (E) Endogenous NUPR1 in MCF10AT cells was silenced by RNA interference. 187-3, -6, -9 denote stable clones that are silenced for NUPR1 using shRNA187. EV denotes empty vector transfectants and SC denotes cells transfected with a non-targeting control shRNA. Loss of NUPR1 is accompanied by a notable change in the levels of p21. (F). Abrogating endogenous NUPR1 increased the sensitivity of the cells to doxorubicin (0.5 lM for 72 h) (⁄p < 0.05). (G) Relative to the MCF10AT cells transfected with empty vector (EV) and non-targeting control shRNA (SC), the NUPR1-silenced cells (shNUPR1) show nuclear localization of p21 (FITC) when assessed by immunocytochemistry. DAPI serves to label the nuclei.
that is concomitant with an increase in P-Thr-145-p21. NUPR1overexpressing cells also showed an overall increase in BCL-xL levels. However, the deactivated phosphorylated 62-serine form of BCL-xL was barely detectable in NUPR1-expressing cells in comparison to vector control cells [28]. We did not detect any change in the P-Ser-146-p21 levels. Phosphorylation of p21 at Ser 146 and/or Thr 145 is associated with cytoplasmic localization of p21 and lead to activated Bcl-xL. In order to assess a role for activated PI-3-K/Akt in mediating phosphorylation of p21 at the Thr145 and/ or Ser146 residues we treated the vector control and NUPR1expressing cells with inhibitors of Akt (Akt Inhibitor IV) and PI-3kinase (wortmannin). We also assessed a role for IKK (NF-jB signaling) using BMS-34551. Treatment with an Akt inhibitor resulted in reduced levels of activated p-Ser-473-Akt with concomitantly decreased levels of p-Thr-145-p21. This was accompanied by an increase in p-Ser-62-Bcl-xL, indicating an increase in the cellular pools of inactive Bcl-XL (Fig. 3 A& B). Notably, while inhibition of NF-jB signaling had no impact on the total cellular pool of p21, inhibition of PI-3-kinase and Akt caused a drastic decrease in the levels of total p21. While not completely resolved, there are a couple of reports documenting a role for Akt in stabilizing p21 [29,30]; and the withdrawal of or interference in Akt signaling destabilizes p21 by targeting it for proteasomal degradation [30]. Inhibition of IKK with BMS-345541 did not result in any notable change in the levels of phosphorylated Akt or p-Thr-145-p21. There was a minimal change in the levels of p-Ser-62-Bcl-xL. Inhibition of PI-3-kinase using wortmannin greatly impacted the activation of Akt as
seen by a complete absence of p-Ser-473-Akt in the treated NUPR1-expressing cells. There was a simultaneous profound reduction in the p-Thr-145-p21 levels. Surprisingly, the pools of p-Ser-62-Bcl-xL remained low in the NUPR1-expressing cells despite wortmannin treatment. The 146-Serine residue of p21 was not affected by any variables imposed in this study. Taken together, these results support our claims that NUPR1 mediates cytoplasmic localization of p21 by phosphorylating its nuclear localization signal in a PI-3 Kinase/Akt dependent manner. In addition, this correlated with subsequent activation of anti-apoptotic Bcl-xL. 4.4. Inhibition of Akt and PI-3K sensitizes NUPR1-expressing cells to doxorubicin In order to understand the determinative role of NUPR1-induced PI-3-K/Akt-mediated phosphorylation and cytoplasmic localization of p21 with the chemoresistance properties of the cells, we assessed the response of the cells to doxorubicin when co-treated with inhibitors of Akt, PI-3-K and IKK. As seen in Fig. 4A and B, while inhibition of Akt and PI-3-kinase restored the sensitivity of the NUPR1-expressing cells to doxorubicin, the NUPR1-expressing cells remained resistant to doxorubicin even in presence of the IKK inhibitor, indicating that the chemoresistance imparted by NUPR1 is independent of the NF-jB pathway (Fig. 4C). Pre-treatment with any of the inhibitors did not impact the response to doxorubicin (data not shown).
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Fig. 2. Blocking PI3K/Akt signaling reverses NUPR1-mediated p21 cytoplasmic localization in SUM159 cells. SUM159 cells were plated on coverslips and treated for 24 h with the indicated inhibitor (Akt Inhibitor IV l lM, or Wortmannin l lM or BMS-345541, l lM). Cells were fixed and stained as indicated in Methods and Materials. Images acquired at 40 magnification. NUPR1 overexpressing cells displayed predominantly cytoplasmic localization of p21, which was reversed by both Akt Inhibitor IV and Wortmannin treatment. Representative images are shown. The ratio of nuclear:cytoplasmic signal of p21 was determined from these images. Mean intensity of p21 staining was determined for each cell in both the cell nucleus and the whole cell. Cytoplasmic intensity was determined indirectly by subtracting the summed nuclear intensity from the summed cellular intensity and controlling for cellular volume. A ratio was then taken for each cell comparing the mean intensity of the cell nucleus to the mean intensity of the cytoplasm. p21 localization becomes similar after treatment with Wortmannin and Akt Inhibitor IV.
Fig. 3. NUPR1 leads to phosphorylation of p21 via intermediates PI-3 Kinase and Akt and is correlated with increased levels of hypophosphorylated BCL-xL. (A) NUPR1 overexpressing SUM159 cells displayed increased total Akt and the active phosphorylated form of Akt (phospho-473-Serine Akt). These cells also showed increased phosphorylation of p21 at the 145-Threonine residue but not at the 146-Serine residue. Treatment for 2 h with Akt and PI-3 Kinase inhibitors (Akt Inhibitor IV and Wortmannin, respectively) reversed the phosphorylation status of both Akt and p21. Inhibition of IKK using BMS-345541 did not impact the phosphorylation of p21. NUPR1 overexpressing SUM159 cells also demonstrated increased levels of BCL-xL but decreased phosphorylated 62-Serine signal. Cells treated with Akt Inhibitor or Wortmannin for 24 h showed an increase in the levels of phosphorylated BCL-xL. (B) Densiometric analysis of p21 phosphorylation status reveals decrease in p21 phosphorylation of the 145Threonine residue after treating NUPR1 overexpressing cells with Akt Inhibitor IV and Wortmannin, but not IKK inhibitor. The levels of Bcl-xL phosphorylation (indicating inactive Bcl-xL) is increased when the NUPR1-expressing cells are treated with Akt inhibitor relative to untreated cells. Wortmannin or BMS-345541 treatment does not impact Bcl-xL phosphorylation.
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Fig. 4. NUPR1 mediates chemoresponsiveness via PI-3-K/Akt. SUM159 cells transfected with empty vector or NUPR1 were treated with doxorubicin (0.5 lM) and/or Akt inhibitor IV (1 lM) or wortmannin (1 lM) or IKK inhibitor (1 lM) and cell viability was assessed 24 h later by MTS assay. The NUPR1 cells demonstrated increased proliferation in culture (untreated). While the inhibitors alone did not impact cell viability, when co-treated with (A) Akt inhibitor or (B) wortmannin, the NUPR1-expressing cells regained sensitivity to doxorubicin. (C) Inhibition of IKK did not impact cellular response to doxorubicin (even when higher concentrations of the IKK inhibitor were used; data not shown).
5. Discussion NUPR1 was first identified as a protein physiologically involved in the cellular stress response, promoting regeneration and cellular growth [31]. NUPR1 functions in the nucleus where it serves as a co-factor for transcription factors such as p53 [9] and estrogen receptor-b [32]. Since NUPR1 is a stress molecule that might be upregulated in actual in vivo conditions, where the tumor cells undergo selection pressure, stress from the surrounding cells and from the host’s defense mechanisms may play critical roles in influencing the activity of NUPR1. The potential of NUPR1 in making cancer cells resistant to the effects of conventional chemotherapeutic agents is suggested by two independent studies examining the effects of NUPR1 expression in pancreatic [33] and breast cancer [9]. Gemcitabine, the drug of choice for pancreatic cancer, was found to target NUPR1. Gemcitabine resistant pancreatic cancer cells devoid of NUPR1 (NUPR1 / ) were more prone to apoptosis than their wild type counterparts, and gemcitabine-induced apoptosis mainly resulted from the inhibition of the constitutive antiapoptotic activity of NUPR1 [3]. Alternatively, named as cyclin-dependent kinase inhibitor-IA (CDKN1A), CDK-interacting protein 1 (CIP1) and wild-type p53activated fragment 1 (WAF1), p21 is a protein possessing duality in the regulation of cancer behavior. The nuclear form of p21 mainly executes its tumor suppressing function by binding to CDK complexes, inhibiting the activity of cyclin-dependent kinases, such as, Cdk2, Cdk3, Cdk4 and Cdk6, leading to cell growth arrest [14]. In contrast, the cytoplasmic form of p21 complexes with and inactivates pro-apoptotic proteins such as caspase-3 [12] and ASK-1 [13,34]. This oncogenic function of p21 has also been validated by clinical evidence. Higher expression of cytoplasmic p21 has been verified as a factor of poor prognosis in human primary breast carcinomas [23,35]. Moreover, resistance to chemotherapeutic drugs may result from accumulation of cytoplasmic p21, as paclitaxel could increase cytoplasmic p21, and reverse the cell cycle inhibitory effect of p21 [25,26]. Thus, cellular localization plays a critical role to determine the function of p21, i.e. nuclear p21, as a cell cycle inhibitor, represses cell growth; cytoplasmic p21, as an anti-apoptotic factor, enhances cell survival. Phosphorylation of p21 at Ser 146 and/or Thr 145 is associated with cytoplasmic localization of p21 [22]. Activated Akt phosphorylates p21 at Ser 146 and stabilizes p21; phosphorylation of p21 at the Thr 145 position makes p21 a positive modulator of cell survival and proliferation. Specifically, phosphorylated p21 does not inhibit cyclin E-CDK2 but instead, promotes the binding and assembly and activation of the cyclin D1-CDK4 complex, which is a critical mediator of G1-S progression, acting epistatic to and inhibiting p27. Phosphorylation of
p21 at Thr145 releases PCNA from p21 binding and resultant inhibition. This release allows for elevated p21 levels to exist in cells without inhibiting the cell’s ability to progress through S phase [30,36]. As such, phosphorylation of p21 at these two critical residues not only alters its sub-cellular localization, but also interferes in the growth-restrictive properties of p21 so profoundly, making it a pro-survival molecule. We have previously shown that p53 and p300 are required for NUPR1 mediated up-regulation of p21 [9]. Mutations in p53 are seen in about 30% of breast cancers. Triple-negative basal-like breast cancers account for 15% of breast cancers and have an increased frequency of p53 mutations. Mutant p53 status is a strongly unfavorable prognostic indicator in these patients for overall survival and response to anthracycline-containing therapy. Inflammatory breast cancer (T4d in the TNM classification) has a particularly aggressive behavior and poor prognosis, and mutations in p53 are more frequent (50%) in inflammatory than in non-inflammatory breast cancer (20–30%) [37]. The SUM159PT cell line, derived from inflammatory breast cancer contains a p53 loss of function missense mutation [18], and is triple-negative for ER, PR, and HER2/Neu [38]. Thus, it presents a unique system to study the role of NUPR1 in chemoresistance. In these cells NUPR1 expression did not result in an appreciable increase in p21, but rather resulted in altered localization of p21. Thus, the regulation of sub-cellular localization of p21 mediated by Akt is, from the perspective of NUPR1-induced chemoresistance, a novel finding. Akt has been well studied for its role in inhibiting apoptosis and promoting cell survival. The PI-3K-Akt pathway has been linked to resistance to a variety of chemotherapeutics, such as gemcitabine, irinotecan, etoposide, and Taxol [39–43]. Hyperphosphorylation of Akt has also been linked to poor prognosis in a variety of cancers [44]. Our results show that p21 that is phosphorylated by Akt is unable to carry out its tumor suppressive functions, and instead causes activation of BCL-xL available in the cellular pool to complex with and de-activate pro-apoptotic Bax. NUPR1 overexpression increases cellular proliferation, while removing necessary cell cycle checkpoints [9], and our findings elucidate a NUPR1PI-3-K/Akt-phospho-p21 axis that enhances chemoresistance in breast cancer cells. Various PI-3-K/Akt inhibitors are in clinical trials. It is possible that the use of such inhibitors may help restore chemosensitivity in patients whose tumors show upregulated NUPR1 expression, thereby expanding the scope of the treatment. 6. Grant support We gratefully acknowledge funding from the Norwegian Radium Research Foundation. This study was performed at the University of South Alabama Mitchell Cancer Institute.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.07. 063. References [1] Hortobagyi, G.N., Heim, W., Hutchins, L., Rivera, E., Mason, B., Booser, D.J. and Kirshner, J. (2010) A phase 2 study of a fixed combination of uracil and ftorafur (UFT) and leucovorin given orally in a 3-times-daily regimen to treat patients with recurrent metastatic breast cancer. Cancer 116, 1440–1445. [2] Castiglione-Gertsch, M., The International Breast Cancer Study Group, et al. (1997) Retreating recurrent breast cancer with the same CMF-containing regimen used as adjuvant therapy. Eur. J. Cancer 33, 2321–2325. [3] Giroux, V., Malicet, C., Barthet, M., Gironella, M., Archange, C., Dagorn, J.C., Vasseur, S. and Iovanna, J.L. (2006) p8 is a new target of gemcitabine in pancreatic cancer cells. Clin. Cancer Res. 12, 235–241. [4] Su, S.B. et al. (2001) Expression of p8 in human pancreatic cancer. Clin. Cancer Res. 7, 309–313. [5] Ree, A.H. et al. (2002) Clinical and cell line specific expression profiles of a human gene identified in experimental central nervous system metastases. Anticancer Res. 22, 1949–1957. [6] Ito, Y. et al. (2003) Expression and cellular localization of p8 protein in thyroid neoplasms. Cancer Lett. 201, 237–244. [7] Mohammad, H.P., Seachrist, D.D., Quirk, C.C. and Nilson, J.H. (2004) Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing hormone. Mol. Endocrinol. 18, 2583–2593. [8] Kong, D.K., Georgescu, S.P., Cano, C., Aronovitz, M.J., Iovanna, J.L., Patten, R.D., Kyriakis, J.M. and Goruppi, S. (2010) Deficiency of the transcriptional regulator p8 results in increased autophagy and apoptosis, and causes impaired heart function. Mol. Biol. Cell 21, 1335–1349. [9] Clark, D.W., Mitra, A., Fillmore, R.A., Jiang, W.G., Samant, R.S., Fodstad, O. and Shevde, L.A. (2008) NUPR1 interacts with p53, transcriptionally regulates p21 and rescues breast epithelial cells from doxorubicin-induced genotoxic stress. Curr. Cancer Drug Targets 8, 421–430. [10] Ree, A.H. et al. (1999) Expression of a novel factor in human breast cancer cells with metastatic potential. Cancer Res. 59, 4675–4680. [11] Sandi, M.J., Hamidi, T., Malicet, C., Cano, C., Loncle, C., Pierres, A., Dagorn, J.C. and Iovanna, J.L. (2011) p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis. J. Cell Physiol. 226, 3442– 3451. [12] Asada, M., Yamada, T., Ichijo, H., Delia, D., Miyazono, K., Fukumuro, K. and Mizutani, 5. (1999) Apoptosis inhibitory activity of cytoplasmic p21(Cip1/ WAF1) in monocyticdifferentiation. EMBO J. 18, 1223–1234. [13] Blagosklonny, M.V. (2002) Are p27 and p21 cytoplasmic oncoproteins? Cell Cycle 1, 391–393. [14] Harper, J.W. et al. (1995) Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell 6, 387–400. [15] Flores-Rozas, H., Kelman, Z., Dean, F.B., Pan, Z.Q., Harper, J.W., Elledge, S.J., O’Donnell, M. and Hurwitz, J. (1994) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc. Natl. Acad. Sci. USA 91, 8655– 8659. [16] Zhan, J., Easton, J.B., Huang, S., Mishra, A., Xiao, L., Lacy, E.R., Kriwacki, R.W. and Houghton, P.J. (2007) Negative regulation of ASK1 by p21Cip1 involves a small domain that includes Serine 98 that is phosphorylated by ASK1 in vivo. Mol. Cell. Biol. 27, 3530–3541. [17] Yamamoto, K., Ichijo, H. and Korsmeyer, S.J. (1999) BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol. Cell. Biol. 19, 8469–8478. [18] Wasielewski, M., Elstrodt, F., Klijn, J.G., Berns, E.M. and Schutte, M. (2006) Thirteen new p53 gene mutants identified among 41 human breast cancer cell lines. Breast Cancer Res. Treat. 99, 97–101. [19] Morrow, K.A., Das, S., Metge, B.J., Ye, K., Mulekar, M.S., Tucker, J.A., Samant, R.S. and Shevde, L.A. (2011) Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation. J. Biol. Chem. 286, 40376– 40385. [20] Vasseur, S., Vidal Mallo, G., Fiedler, F., Bodeker, H., Canepa, E., Moreno, S. and Iovanna, J.L. (1999) Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur. J. Biochem. 259, 670–675. [21] Jiang, Y.F., Vaccaro, M.I., Fiedler, F., Calvo, E.L. and Iovanna, J.L. (1999) Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro. Biochem. Biophys. Res. Commun. 260, 686–690.
[22] Zhou, B.P., Liao, Y., Xia, W., Spohn, B., Lee, M.H. and Hung, M.C. (2001) Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER- 2/neu-overexpressing cells. Nat. Cell. Biol. 3, 245–252. [23] Xia, W., Chen, J.S., Zhou, X., Sun, P.R., Lee, D.F., Liao, Y., Zhou, B.P. and Hung, M.C. (2004) Phosphorylation/cytoplasmic localization of p21Cip1/WAF1 is associated with HER2/neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients. Clin. Cancer Res. 10, 3815–3824. [24] Yuste, L., Montero, J.C., Esparis-Ogando, A. and Pandiella, A. (2005) Activation of ErbB2 by overexpression or by transmembrane neuregulin results in differential signaling and sensitivity to herceptin. Cancer Res. 65, 6801–6810. [25] Perez-Tenorio, G., Berglund, F., Esguerra Merca, A., Nordenskjold, B., Rutqvist, L.E., Skoog, L. and Stal, O. (2006) Cytoplasmic p21WAF1/CIP1 correlates with Akt activation and poor response to tamoxifen in breast cancer. Int. J. Oncol. 28, 1031–1042. [26] Heliez, C., Baricault, L., Barboule, N. and Valette, A. (2003) Paclitaxel increases p21 synthesis and accumulation of its AKT-phosphorylated form in the cytoplasm of cancer cells. Oncogene 22, 3260–3268. [27] Ping, B. et al. (2006) Cytoplasmic expression of p21CIP1/WAF1 is correlated with IKKbeta overexpression in human breast cancers. Int. J. Oncol. 29, 1103– 1110. [28] Upreti, M., Galitovskaya, E.N., Chu, R., Tackett, A.J., Terrano, D.T., Granell, S. and Chambers, T.C. (2008) Identification of the major phosphorylation site in BclxL induced by microtubule inhibitors and analysis of its functional significance. J. Biol. Chem. 283, 35517–35525. [29] Park, J.K., Cho, C.H., Ramachandran, S., Shin, S.J., Kwon, S.H., Kwon, S.Y. and Cha, S.D. (2006) Augmentation of sodium butyrate-induced apoptosis by phosphatidylinositol 3-kinase inhibition in the human cervical cancer cellline. Cancer Res. Treat. 38, 112–117. [30] Li, Y., Dowbenko, D. and Lasky, L.A. (2002) AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277, 11352–11361. [31] Mallo, G.V., Fiedler, F., Calvo, E.L., Ortiz, E.M., Vasseur, S., Keim, V., Morisset, J. and Iovanna, J.L. (1997) Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth. J. Biol. Chem. 272, 32360–32369. [32] Jiang, W.G., Davies, G. and Fodstad, O. (2005) Com-1/P8 in oestrogen regulated growth of breast cancer cells, the ER-beta connection. Biochem. Biophys. Res. Commun. 330, 253–262. [33] Vasseur, S. et al. (2004) p8 improves pancreatic response to acute pancreatitis by enhancing the expression of the anti-inflammatory protein pancreatitisassociated protein I. J. Biol. Chem. 279, 7199–7207. [34] Suzuki, A., Tsutomi, Y., Akahane, K., Araki, T. and Miura, M. (1998) Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene 17, 931–939. [35] Winters, Z.E., Hunt, N.C., Bradburn, M.J., Royds, J.A., Turley, H., Harris, A.L. and Norbury, C.J. (2001) Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/ CIP1) in breast cancer association with prognosis. Eur. J. Cancer 37, 2405– 2412. [36] Malumbres, M. and Barbacid, M. (2006) Is Cyclin D1-CDK4 kinase a bona fide cancer target? Cancer Cell 9, 2–4. [37] Varna, M., Bousquet, G., Plassa, L.F., Bertheau, P. and Janin, A. (2011) TP53 status and response to treatment in breast cancers. J. Biomed. Biotechnol., 284584. [38] Hollestelle, A. et al. (2010) Distinct gene mutation profiles among luminaltype and basal-type breast cancer cell lines. Breast Cancer Res. Treat. 121, 53– 64. [39] Campbell, R.A., Bhat-Nakshatri, P., Patel, N.M., Constantinidou, D., Ali, S. and Nakshatri, H. (2001) Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J. Biol. Chem. 276, 9817–9824. [40] Perez-Tenorio, G. and Stal, O. (2002) Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br. J. Cancer 86, 540–545. [41] Shah, A. et al. (2005) Phospho-akt expression is associated with a favorable outcome in non-small cell lung cancer. Clin. Cancer Res. 11, 2930–2936. [42] Bleau, A.M., Hambardzumyan, D., Ozawa, T., Fomchenko, E.I., Huse, J.T., Brennan, C.W. and Holland, E.C. (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 4, 226–235. [43] Sinnberg, T. et al. (2009) Inhibition of PI3K-AKT-mTOR signaling sensitizes melanoma cells to cisplatin and temozolomide. J. Invest. Dermatol. 129, 1500– 1515. [44] Cheng, J.Q., Lindsley, C.W., Cheng, G.Z., Yang, H. and Nicosia, S.V. (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24, 7482–7492.