PKCδ-dependent Activation of the Ubiquitin Proteasome System is Responsible for High Glucose-induced Human Breast Cancer MCF-7 Cell Proliferation, Migration and Invasion

Breast cancer is one of the most common cancers and is the most common cause of cancer mortality in women worldwide (Jemal et al., 2011). Several type 2 diabetes mellitus (T2DM) risk factors, such as old age and obesity, are associated with breast cancer (Wolf et al., 2005). Conversely, T2DM is linked to increasing breast cancer incidence and mortality (Liao et al., 2011). Meta-analysis reveals that breast cancer risk in patients with T2DM is increased by 20-40% (Larsson et al., 2007). Diabetic women have a higher risk of dying from breast cancer than those without diabetes (Coughlin et al., 2004). One possible mechanism is that the hyperglycemia in T2DM patients may promote cancer progression, because the neoplastic cells could use glucose for proliferation via the pentose phosphate pathway (Warburg, 1956; Dang and Semenza, 1999). However, the detailed mechanism(s) remains unknown. The ubiquitin proteasome system (UPS) and the autophagy-lysosome pathway are two main routes of the intracellular protein-degradation (Goldberg, 2003; Rubinsztein, 2006). Current studies demonstrate that


Introduction
impaired protein degradation promotes the pathogenesis of some diseases including cancers. UPS deregulation plays a key role in cell cycle progression, proliferation, apoptosis, angiogenesis and cell signaling pathways that contribute to cancer development (Ciechanover and Schwartz, 1994;Hochstrasser, 1995;Ciechanover et al., 2000;Orlowski and Dees, 2003). Thus, it is necessary to keep the UPS functional for normal and neoplastic cell survival. It is interesting that diabetes increases ubiquitin expression but does not alter proteasome chymotrypsinlike peptidase activity in the heart and skeletal muscle (Liu et al., 2000). We do not know whether the UPS is involved in high glucose-induced carcinogenesis regulation.
In the present study, we determined that high glucoseinduced proliferation, migration, and invasion were attenuated by treatment with the UPS inhibitor bortezomib (BZM) in human breast cancer MCF-7 cells. We also demonstrated that both the PKCδ specific inhibi¬tor rottlerin and PKCδshRNA suppressed UPS activity. Our data suggest that PKCδ mediated high glucose-induced UPS activation and that targeting the UPS and reducing serum glucose levels may represent a novel therapeutic strategy for breast cancer patients.

Proliferation assay
The MTT assay was used to evaluate cell proliferation. The thiazolyl blue tetrazolium bromide (MTT) (Amresco, Solon, OH, USA) was dissolved in phosphate buffered saline(PBS) at a concentration of 5 mg/ml, filtered, and stored at 4 °C. Cells were seeded into a 96-well plate, washed three times with PBS and cultured in RPMI 1640 without FBS and glucose. After 12 hours, cells were treated with the indicated glucose concentrations of (Sigma, St. Louis, MO, USA), bortezomib (BZM, Santa Cruz, Texas, USA) and/or rottlerin (Sigma) for 48 hrs. The osmotic pressure was balanced by D-Mannitol (Sigma). For the proliferation assay, 20 μl MTT was added into each well. An ELISA plate reader (Biotek, Winooski, Vermont, USA) was used to measure the optical density at 490 nm.

TUNEL assay
The TUNEL assay was performed using an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) following the manufacturer's instructions. Cells that were cultured in RPMI 1640 containing 10% FBS were used as a negative control. Images were taken with the Olympus FluoView FV1000 Confocal Microscope.
Wound healing assay MCF-7 cells were grown to confluent monolayers on 6-well plates and a pipette tip was used to create linear scratch wounds. Mitomycin C (Amresco) was used to inhibit cell proliferation. Wound images were taken with a digital camera mounted on light microscope. The wound gap widths were measured using Image J software.

Transwell assay
The upper chamber of each 8.0 µm pore size Transwell apparatus (Corning, NY, USA) was coated with Matrigel (BD Biosciences, San Jose,CA). MCF-7 cells were added to the upper chamber at a density of 10 6 cells/ml (100 µl per chamber) and incubated for 24 hrs followed by removal of the cells that remained in the top chamber with cotton swabs. Cells that penetrated to the lower membrane surface were fixed in 4% paraformaldehyde, stained with crystal violet, and counted under a microscope.

Plasmids and virus infection
PKCδ shRNA lentiviruses were obtained from Santa Cruz Biotechnology, RFP adenoviruses and specific ubiquitin-proteasome reporter GFPu were kind gifts from Dr. Xuejun (XJ) Wang (The University of South Dakota). MCF-7 cells were infected with PKCδ shRNA lentiviruses (MOI: 50), GFPu adenoviruses (MOI: 20), or RFP adenoviruses (MOI: 20) for 6 hrs and were then cultured in RPMI 1640 containing 5.5 mM glucose for 24 hrs for experiments. Cell lysates were collected and Western blots were performed to detect protein expression using specific antibodies.

Western blots
Cells were collected with lysis buffer after being washed three times with ice-cold PBS. Lysates were boiled in SDS loading buffer for 10 min then cleared by centrifugation (14,000 rpm, 10min, 4 °C). The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with specific antibodies.

Statistical analysis
Results are represented as the mean ± SEM. The quantification of the relative increase in protein expression and phosphorylation was performed using NIH Scion Image software and was normalized with the control protein expression in each experiment. The figures were representative of at least three independent experiments with similar results. Differences between mean values were examined using the paired Student's t-test. p < 0.05 was considered to be statistically significant.

Effects of high glucose on MCF-7 cell growth, migration, and invasion
To investigate the effects of high glucose on cancer cell growth, MCF-7 cells were incubated in 5.5 mM, 11 mM, or 22 mM glucose for 48 hrs. MTT and TUNEL assays were performed to measure cell proliferation and apoptosis. As demonstrated in Figure 1A, glucose enhanced MCF-7 cell proliferation in a concentration-dependent manner. Accordingly, high glucose suppressed MCF-7 cell apoptosis ( Figure 1B). We then studied the effects of high glucose on MCF-7 cell migration and invasion using wound healing and transwell assays as described in the materials and methods section. Mitomycin C was administered to inhibit cell proliferation. We determined that relative wound closure ( Figure 1C) and the number of invading cells ( Figure 1D) was significantly increased in cells that had been treated with 11 mM and 22 mM glucose for 48 hrs.

Effects of the ubiquitin-proteasome system (UPS) activity on high glucose-treated MCF-7 cell growth, migration, and invasion
During the past decade, it has been suggested that the ubiquitin-proteasome system is involved in cancer development, and targeting this pathway with proteasome inhibitors such as bortezomib (BZM) is a potential strategy for cancer treatment (Dees and Orlowski, 2006;Landis-Piwowar et al., 2006;Sato et al., 2008). To figure out whether glucose regulated UPS activity, we first assessed proteasome peptidase activity in MCF-7 cells that had been treated with the indicated glucose concentrations for 48 hrs. As demonstrated in Figure 2A, chymotrypsinlike peptidase activity was increased in a glucose concentration-dependent manner. However, trypsin-like and caspase-like proteasome peptidase activities remained unchanged (data not shown). To confirm that the UPS was specifically activated by high glucose, MCF-7 cells were coinfected with RFP (MOI: 20) and GFPu adenoviruses (MOI: 20) followed by incubation in the presence of 5.5, 11, or 22 mM glucose for 48 hrs. Western blots were performed to detect RFP and GFPu protein expression. We determined that high glucose significantly decreased GFPu protein expression and the GFPu to RFP ratio ( Figure 2B). To determine the effects of UPS on high glucose-induced cancer cell growth, migration, and invasion, MCF-7 cells were pretreated with 50 nM BZM for 1 hr followed by incubation in the presence of 11 or 22 mM glucose for another 48 hrs. We determined that BZM pretreatment significantly mitigated high glucose-induced cell growth ( Figure 2C), enhanced apoptosis ( Figure 2D), suppressed wound closure ( Figure 2E) and reduced invasion of MCF-7 cells ( Figure 2F).

Effects of PKCδ on UPS activity in high glucose-treated MCF-7 cells
To elucidate the mechanism by which UPS activity was upregulated, MCF-7 cells were cultured in RPMI 1640 containing 5.5, 11, or 22 mM glucosefor 48 hrs. PKC isoform phosphorylation was detected by Western blot using specific antibodies. Our data demonstrated that PKCδ phosphorylation was significantly enhanced by high glucose treatment ( Figure 3A). Other PKC isoform phosphorylation remained unchanged (data not shown). To investigate the effect of PKCδ on UPS activity, MCF-7 cells were pretreated with the PKCδ specific inhibitor rottlerin (250 nM) for 1 hr followed by treatment with 11 or 22 mM high glucose for another 48 hrs, and proteasomal peptidase activity was measured. As demonstrated in Figure 3B, rottlerin pretreatment significantly decreased chymotrypsin-like peptidase activity. To confirm the effects of PKCδ on the UPS, we investigated whether PKCδ activity affected specific proteasome substrate GFPu expression. RFP and GFPu were overexpressed in MCF-7 cells by coinfecting cells with RFP and GFPu adenoviruses (MOI: 20). PKCδ was inhibited by pretreating the cells with rottlerin (250 nM) for 1 hr. PKCδ was knocked down in MCF-7 cells by PKCδ shRNA lentivirus infection (MOI: 100). Cells were then incubated in the presence of 5.5 or 22 mM glucose for another 48 hrs. We determined that both rottlerin ( Figure  3C) and PKCδ shRNA ( Figure 3D) restored GFPu protein expression and the GFPu to RFP ratio.

Effects of PKCδ inhibition on high glucose-treated MCF-7 cell growth, migration, and invasion
To confirm the functional consequences of PKCδ inhibition on cancer cell growth, migration, and invasion, MCF-7 cells were incubated with 11 or 22 mM glucose for 48 hrs. Cell growth, migration, and invasion were assessed by MTT, TUNEL, wound healing, and transwell assays, respectively. The results demonstrated that the PKCδ inhibitor rottlerin significantly reduced cell proliferation ( Figure 4A), increased apoptosis ( Figure 4B), suppressed wound-closure ( Figure 4C) and reduced the number of invading cells ( Figure 4D).

Discussion
In recent years, type 2 diabetes mellitus (T2DM) has been suggested to be a negative prognostic factor for breast cancer, especially in premenopausal women (Liao et al., 2010). One potential mechanism for this correlation is that hyperglycemia or high glucose benefits advanced tumor propagation via the Warburg effect (Hauptmann et al., 2005;Gillies and Gatenby, 2007;Kroemer and Pouyssegur, 2008). Our finding in the present study revealed that high glucose significantly increased human breast cancer MCF-7 cell growth, migration, and invasion, which supports this theory (Figure 1).
The UPS is a main route for protein degradation that plays an important role in maintaining protein homeostasis and quality control. Previous studies have demonstrated that the UPS is involved in regulating some very important cellular processes such as signal transduction, growth, proliferation, differentiation and apoptosis. Abnormal UPS function can result in a malignant cellular phenotype. Thus, proteasome inhibition is an attractive target for novel anticancer therapy (Marfella et al., 2007;Wu et al., 2010;Bedford et al., 2011;Driscoll and Woodle, 2012;Micel et al., 2013). Although the role of the UPS in type 2 diabetes pathogenesis remains largely unexplored, some studies have suggested that UPS activation is responsible for the diabetes-induced insulin signaling impairment, pancreaticbeta-cell dysfunction, and cardiovascular complications (Wing, 2008;Marfella et al., 2009;Kirk-Ballard et al., 2013). In contrast, Liu et al. demonstrated that acute diabetes did not increase proteasome chymotrypsin-like peptidase activity in the heart and skeletal muscle (Liu et al., 2000). Selected UPS genes and proteasome activity were downregulated in T2D islets and beta cell fractions (Bugliani et al., 2013). These data highlight the importance of the UPS in cancer and diabetes development. In the present study, we determined that chymotrypsin-like peptidase activity was significantly increased in high glucose-treated MCF-7 cells (Figure 2A). Consistent with this result, GFPu protein expression was reduced ( Figure 2B). Because GFPu is a specific ubiquitin proteasome system substrate that could be used as a dynamic reporter of UPS activity in vivo (Bence et al., 2001), our results gave evidence to support the possibility that high glucose enhanced breast cancer cell UPS activity. Our results also determined that proteasome inhibitor bortezomib (Cvek and Dvorak, 2011) pretreatment significantly mitigated cell proliferation ( Figure 2C), increased apoptosis ( Figure 2D), attenuated would closure ( Figure 2E) and reduced the number of invading cells ( Figure 2F), suggesting that high glucoseinduced MCF-7 cell growth, migration, and invasion are mediated by the UPS.
The remaining question is how does high glucose activate the UPS? We determined that high glucose specifically induced PKCδ phosphorylation ( Figure  3A). This finding is consistent with previous studiesthat demonstrated that high glucose activated PKCs. A possible mechanism for inducing PKCs activation is increased reactive oxygen species (ROS), which are generated in response to hyperglycemia (Srivastava, 2002;Wu, 2006;Sasaki and Inoguchi, 2012). Our results also demonstrated that specific PKCδ inhibitor rottlerin pretreatment significantly suppressed high glucoseinduced chymotrypsin-like peptidase activity ( Figure 3B). The high glucose-mediated GFPu expression reduction was restored by both rottlerin administration and PKCδ knockdown ( Figure 3C and Figure 3D). These findings strongly suggest that activated PKCδ contributes to  (Smith et al., 2004). PKCs are crucial regulators of cancer cell growth, migration, and invasion (Masur et al., 2011). Our data revealed that rottlerin-mediated PKCδ inhibition suppressed MCF-7 growth, migration, and invasion (Figure 4), consistent with earlier studies demonstrating that PKCδ can enhance malignant mammary cell proliferation, metastasis and survival (Kiley et al., 1999;Grossoni et al., 2007).
In summary, our results demonstrated for the first time that PKCδ-dependent UPS activation upregulated MCF-7 cell growth and invasion after high glucose treatment. Characterizing the mechanism by which PKCδ regulated the UPS and its pathophysiological consequences will provide important information regarding breast cancer diagnosis and therapy.