Protein kinase C δ -dependent regulation of Ubiquitin-proteasome system function in breast cancer

Abstract

Besides the crucial role of hyperinsulinemia in the development of breast cancer with Type 2 diabetes mellitus (T2DM), it has been shown that hyperglycemia could contribute to promote cancer progression. A remarkable association within hyperglycemia, PKC $\delta$ and Ubiquitin-proteasome system (UPS) has been reported, suggesting that PKC $\delta$ may mediate high glucose-induced UPS activation in breast cancer cells. Although the independent effects of PKC $\delta$ or UPS on breast cancer and T2DM are increasingly supported by experimental evidence, the complex interactional link between PKC $\delta$ and UPS is still unclear. Hence, we focus on the relationship between PKC $\delta$ and UPS in breast cancer with T2DM. We hypothesize that PKC $\delta$ may have the function to regulate the activity of UPS. Further, we speculate that PKC $\delta$ combine with proteasome $\alpha$ 2 promoter, that indicate PKC $\delta$ regulate the function of UPS by change the composition of proteasome. Therefore, we surmise that PKC $\delta$ mediated high glucose-induced UPS activation in breast cancer cells, and specific PKC $\delta$ inhibitor rottlerin significantly suppressed elevated glucose induced the activity of UPS. We hope that our paper will stimulate further studies the relationship between PKC $\delta$ and UPS, and a new targeted therapy and early medical intervention for PKC $\delta$ could be a useful option for breast cancer cases complicated with T2DM or hyperglycemia.

1. Introduction

Breast cancer is the most commonly cancer for women worldwide and it is the underlying cause of cancer-related death in women [1]. Type 2 diabetes mellitus (T2DM) is characterized by a chronic hyperglycemic state concurrent with insulin resistance and/or impaired insulin secretion [2]. It now demonstrated that T2DM is related to increase breast cancer incidence and mortality [3, 4, 5, 6, 7]. Indeed, prediabetes may also increase the risk of breast cancer [5, 8, 9, 10, 11]. The detailed mechanisms remain unknown; however, hyperglycemia in T2DM patients may play important roles in the process of breast cancer.

In this paper, we hypothesize that PKC $\delta$ may have the function to regulate the activity of UPS. Further, PKC $\delta$ combine with proteasome $\alpha$ 2 promoter, that indicate PKC $\delta$ regulate the function of UPS by change the composition of proteasome. Therefore, PKC $\delta$ regulated high glucose-induced UPS activation in breast cancer cells, and rottlerin, specific PKC $\delta$ inhibitor, dramatically suppressed high glucose-induced the activation of UPS.

2. The Protein kinase C family

The Protein kinase C (PKC) pathways represent one of the most familiar mechanisms by a signal relay of extracellular response as a phospholipid or calcium-dependent protein kinase [12]. Presence of a cell and tissue changes in the distribution and abundance, which is divided into three sub-categories represent 10 different mammalian PKC isoforms: the “classical” (PKC $\alpha$ , $\beta$ I, $\beta$ II, $\gamma$ ), which may be calcium and diacylglycerol stimulation (DAG) or phorbol esters; “novel” (PKC $\delta$ , $\epsilon$ , $\theta$ , $\eta$ ), which can be activated by DAG, but independent of calcium or phorbol ester; and the ‘atypical’ (protein kinase C $\zeta$ and $\lambda$ / $\iota$ ), which is in response to calcium, DAG and phorbol esters. PKC isoforms are crucial for differentiation, proliferation and apoptosis, but more limited portion of the key [13, 14]. Because of its contribution to intracellular signal transduction, it makes sense to suppose that the alterative expression or activity levels of these kinases may result in diseases, including breast cancer. One of the PKCs identified to exhibit equivocal properties is PKC $\delta$ [15]. Most researches reached consensus on the PKC $\delta$ as a tumor suppressing activity having an anti-proliferative in different cells [15]. While several reports indicated PKC $\delta$ was identified as the pro-proliferative role and a cancer-promoting gene [16].

3. Role of PKC $𝜹$ in breast cancer

The effects of PKC $\delta$ in breast cancer are still vague, and the expression levels of PKC $\delta$ in primary breast cancer are inadequate. Though it was not a prerequisite for the cancer progression, some researches have confirmed that overexpressing PKC $\delta$ is able to be a tumor promotion in breast cancer cells [17, 18]. PKC $\delta$ is the most important partner in Ras/Raf/MEK/ERK pathway, that has been proved to accelerate cellular proliferation and tumorigenesis [19]. Insulin growth factor-I (IGF-I) signaling is another primary signaling in regulating growth. Interplays between PKC $\delta$ and mTOR have demonstrated for modulating stress and IGF-I targeted the Insulin receptor substrate 1 (IRS-1) phosphorylation in MCF-7 breast cancer cells [20]. On the other hand, some literatures have confirmed that PKC $\delta$ was response to anti-proliferative effect. For instance, the inositol hexaphosphate possessed the antimitogenic effect in MCF-7 by PKC $\delta$ -induced phosphorylation inhibition of ERK, AKT and Rb [21].

PKC $\delta$ also plays an ambiguous role in cell cycle modulation and apoptosis. It has revealed that PKC $\delta$ plays a proapoptotic role in MCF-7 for UV damage by a caspase dependent mechanism [22]. This PKC $\delta$ -activated mechanism results in the phosphorylation of aSMase, leading to apoptosis ceramide generation and signal enhancement [23]. In contrast, PKC $\delta$ has anti-apoptotic effect in MDA-MB-231and MCF-7 for ionizing radiation [24] and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis [25]. In addition, Grossini et al. [26] has shown that PKC $\delta$ promotes proliferation in murine mammary cells. PKC $\delta$ regulates the G1/S phase transition to influence cell cycle. PKC $\delta$ mediates p21-dependent pathway in SKBR-3 and p27 ${}^{\text{Kip1}}$ -dependent pathway in MCF-7 resulting in G1 arrest [21, 27, 28]. Both the p21 and p27 ${}^{\text{Kip1}}$ can combine with cyclin E/Cdk2 complex and inhibit its activity.

To meet invasion and metastasis of tumor, they must be capable of progression. PKC $\delta$ has been demonstrated to inhibit the MCF-7migration [29]. This reduction may be partly due to the regulation of PKC $\alpha$ . However, for breast cancer cells PKC $\delta$ has been shown to activate MMP-9 [30, 31], again increasing the ambiguity of PKC $\delta$ effects on breast cancer progress.

Numerous studies show that the crosstalk between PKC $\delta$ and Estrogen receptor (ER). Inhibition of PKC $\delta$ blocks most of the 17-B-estriadiol-regulated but not affects TNF $\alpha$ - activated ERK1/2 in MCF-7 [32]. Markov et al. [33] have reported that the methylation of PKC $\delta$ gene in ER+ breast cancer is correlated with a favorable response to anti-estrogenic therapy. However, another study [34] found that raised PKC $\delta$ protein is related to the sensitivity of endocrine therapy. In addition, tamoxifen-resistant breast cancer cells usually express higher PKC $\delta$ protein than their parental tamoxifen-sensitive cells [34, 35]. Regarding a potential role for PKC $\delta$ as a target for breast cancer therapy, AD198, a new doxorubicin analogues lacking the ability to bind and inhibit DNA topoisomerase II, induces apoptosis by activating PKC $\delta$ [36].

PrognoScan is a meta-analysis database served as the prognostic value of genes [37]. PrognoScan is publicly acquired at http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html. The meta-analysis was carried out using the Comprehensive meta-analysis system version 2 (Comprehensive Meta-Analysis; Biostat Inc, Englewood, NJ). The results clearly indicate that PKC $\delta$ expression was non-significant trend toward worse OS, but PKC $\delta$ overexpression was significantly associated with a lower rate of DFS, as shown in Fig. 1 (OS, RR $=$ 1.065, P $=$ 0.497; DFS, RR $=$ 1.895, P $<$ 0.001). These finding further confirm that PKC $\delta$ was potential biomarkers and associated with poor prognosis of breast cancer.

Figure 1.

The expressions of PKC $\delta$ correlate with overall survival (OS) and disease free survival (DFS)in breast cancer patients. (A). The forest plot curves showed correlation between OS and PKC $\delta$ expression levels in breast cancer patients. (B). The forest plot curves showed correlation between DFS and PKC $\delta$ expression levels in breast cancer patients.

4. Role of PKC $𝜹$ in diabetes

Type 2 diabetes mellitus (T2DM) and obesity are main global health problem, and the incidence is expected to increase further in the coming two decades. Genome-wide scanning found out the difference in the development of diabetes by several Quantitative trait locus (QTLs) [38, 39]. The chromosome 14 is the strongest QTL and is related to the occurrence of hyperinsulinemia and insulin resistance. Gene screening associated with insulin resistance in a region is PRKCD encoding PKC $\delta$ . PKC $\delta$ played an important effect in insulin resistance by fatty acid and lipogenesis in liver [40, 41], PKC $\delta$ has also primary modulation effect of insulin resistance [42]. PKC $\delta$ regulates muscle glucose uptake [43], lipogenesis [41], endoplasmic reticulum (ER) stress [44], and angiopoiesis [45]. PKC $\delta$ also affects insulin sensitivity by regulating inflammation. For instance, PKC $\delta$ acts as the regulator in response to TNF- $\alpha$ -induced the activation of NF- $\kappa$ B and ER stress in liver [44]. PKC $\delta$ has also resulted in ROS accumulation of adipocytes in High fat diet (HFD)-treated animals, it could aggravate insulin resistance and increase risk of T2DM [46, 47]. PKC $\delta$ is also resulted in inflammation of adipose tissue, such as PKC $\delta$ activation has leaded to the secretion and production of IL-6 in mesenteric adipose [48]. Therefore, PKC $\delta$ represents a strongly potential therapeutic targets for insulin resistance, T2DM as well as related complications.

5. The ubiquitin proteasome system and breast cancer

The Ubiquitin-proteasome system (UPS) facilitates the degradation of proteins and regulation of growth and stress response and is required for many biological processes such as efficient cell cycle control, stress response, DNA repair, and differentiation [49]. The UPS consists of a variety of crucial enzymes including Ebiquitin-activating enzyme (E1), Ebiquitin-conjugating enzyme (E2), Ebiquitin ligase (E3), the 26S proteasome and the Deubiquitinating enzymes (DUBs) [50, 51]. Normally, E1 activates ubiquitin in an ATP-dependent manner. The activated ubiquitin is then transferred from an E1 enzyme to an E2 enzyme. E3 transfers the activated ubiquitin to lysine residues of targeted proteins, forming a polyubiquitin chain that earmarks the targeted proteins. The specificity of the E3 enzymes determines the specific recognition of target proteins, providing selectivity in which proteins are targeted to the proteasome for degradation [52]. DUBs are a cluster of enzymes that oppose the action of the E3 ligases by cleaving the isopeptide bonds between lysine residues of targeted proteins and the C-terminal glycine of ubiquitin [53, 54].

Tumorigenesis is due to the malfunction in cell growth, proliferation and apoptosis [55]. UPS regulates protein degradation that is involved in cell proliferation, apoptosis, migration, and invasion. Disruption of the ubiquitination and deubiquitination by mutations or modified expression of specific components within the cascade plays an important role in tumorigenesis. Recently, various studies have been conducted to clarify and highlight the roles of UPS in breast cancer.

6. E2 enzymes and breast cancer

Various E2 enzymes such as Ubiquitin-conjugating enzyme 9 (UBC 9), UBC13, UBE2C, E2D3, E2Q1, and E2S have been shown to be involved in the development and progression of breast cancer. UBC9 has been frequently reported to be over-expressed in breast cancer which is positively related with poor breast cancer specific survival [56, 57]. Chen et al. also demonstrated that high Ubc9 expression was associated with poor response to chemotherapy and poor clinical prognosis [58]. UBC13 expression is elevated in primary and metastatic breast cancer and could promote metastatic spread of breast cancer [59]. UBE2C contributes to ubiquitin-mediated proteasome degradation of cell cycle progression in breast cancer [60]. Knockdown or inhibition of UBE2C could reduce proliferation and sensitizes breast cancer cells to radiation, doxorubicin, tamoxifen, and letrozole [61, 62]. UBE2D3 participates in the ubiquitination and degradation of cyclin D1 which is stabilized after UBE2D3 suppression and promotes proliferation of breast cancer cells in vitro [63]. UBE2Q1 could mediate ubiquitination and subsequent proteasomal degradation of p53 [64]. UBE2S is associated with malignant characteristics of breast cancer cells such as migration and invasion [65]. These findings strongly suggest that E2 enzymes are implicated in the development and metastasis of breast cancer.

7. E3 enzymes and breast cancer

There are various E3 enzymes participating in the development of breast cancer, some of which are involved in cancer progression and metastasis. RNF31 highly expressed in breast cancer facilitates p53 polyubiquitination and degradation and promotes cancer progression [66, 67]. The Carboxyl terminus of the Hsc70-interacting protein (CHIP) could result in downregulation of profilin1 and enhanced cell migration [68]. However, another study focusing on ER-positive breast cancer patients has demonstrated that the overexpression of CHIP is a potent prognostic factor of a good prognosis [69]. Further study is needed to clarify this issue. RNF115 (also known as BCA2) is also reported to be overexpressed in ER-positive breast tumors and to promote breast cell proliferation through downregulating the expression of p21 [70]. Besides acting as cancer promoters, some E3 enzymes act as tumor suppressors in breast cancer. HRD1 could suppress the growth and metastasis of breast cancer cells by promoting IGF-1R degradation and attenuated IL-6-induced epithelial-mesenchymal transition [71]. Smurf2 could regulate cell polarity, migration, division, differentiation, and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling, and transcription. Recently, Liu et al. found that Smurf2 might serve as a tumor suppressor in triple negative breast cancer [72]. E6AP, acting both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors could suppress breast cancer invasiveness, colonization, and metastasis by regulating actin cytoskeletal remodeling via regulation of Rho GTPases [73]. There are still many E3 enzymes playing important roles in breast cancer development, progression and metastasis. We thus summarized several recently reported E3 enzymes in Table 1.

Table 1

The studies on E3 enzymes and breast cancer

E3	Targeted protein	Biological process	Reference
HRD1	IGF1R	Cell growth, invasion and migration	[71]
RNF31	P53/ERa	Cell death and proliferation	[66, 67]
Cbl-b	RANK	Cell migration	[74]
Siah	Zeb1	Epithelial to mesenchymal transition	[75]
RNF20/40	Eg5	Cell cycle	[76]
RNF115	p21Waf/Cip1	Cell growth and proliferation	[70]
UBE3A	Annexin A2	Proliferation and apoptosis	[77]
Smurf2	CNKSR2	Cell polarity, migration, division, differentiation	[72]
CHIP	Profilin1	Cell migration	[68, 69]
PIRH2	P53, c-Myc	Proliferation	[78, 79]
Cullin7	P53	promotes cell proliferation and invasion	[80]
E6AP	ECT2-Rho	Represses metastasis	[73]
EDD	P53	Cancer progression	[81]
HACE1	Rac1	Cancer progression	[82]

8. DUBs and breast cancer

DUBs play important roles in ubiquitin processing, reversal of ubiquitin signaling and recycling of ubiquitin. There are about 100 DUBs in human body categorized into six classes: Ubiquitin-specific proteases (USPs), ubiquitin carboxy-terminal hydrolases (UCHs), Ovarian-tumor proteases (OTUs), Machado-Joseph disease protein domain proteases, JAMM/MPN domain-associated metallopeptidases (JAMMs) and Monocyte chemotactic protein-induced protein (MCPIP) family [83, 84]. These DUBs also play crucial roles in breast cancer. ATXN3L, a member of Machado-Joseph disease protein domain proteases, could promote breast cancer proliferation by deubiquitinating Krüppel-like factor 5 [85]. USP2 which belongs to USPs was overexpressed in triple negative breast cancer and associated with increased cell migration and invasiveness [86]. However, some DUBs play a controversial role in breast cancer. For example, Li et al. found that the USP4 could inhibit breast cancer cell growth while other studies demonstrated USP4 could stimulate the TGF $\beta$ -mediated EMT, invasion and metastasis [87].

9. PKC $𝜹$ and the Ubiquitin-proteasome system

The remaining question is whether and how does PKC $\delta$ regulate the UPS? Some studies have suggested that PKC $\alpha$ and PKC $\delta$ activation regulates transcriptional activity and degradation of progesterone receptor (PR) by the 26S proteasome [88]. As previously mentioned, our study suggests that the expressions of PKC $\delta$ and its phosphorylation are dramatically increased in breast cancer tissues with diabetes when compared with breast cancer alone. While proteasome chymotrypsin-like activity is also markedly increased, and there is a high degree of correlation between the expression of PKC $\delta$ and chymotrypsin-like protease activities. As a consequence, we speculate that PKC $\delta$ may have the function to regulate the activity of UPS. Further, we have demonstrated that PKC $\delta$ mediates high glucose-induced UPS activation, and specific PKC $\delta$ inhibitor rottlerin significantly suppresses high glucose induced the activity of UPS. This presents a very interesting experimental model through which the interaction of PKC $\delta$ and UPS could be investigated by ChIP assay [17]. In MCF-7 cells, PKC $\delta$ combines with proteasome $\alpha$ 2 promoter, which indicate that PKC $\delta$ regulates the function of UPS by alter the composition of proteasome.

Cytoscape is an open source software project for integrating bimolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework [89]. The results distinctly indicate that c-Myc interacts with PKC $\delta$ and proteasome $\alpha$ 2 promoter for glycometabolism pathways. The results present that PKC $\delta$ connected with c-Myc translation and proteasome $\alpha$ 2 promoter has an affinity with c-Myc. c-Myc is a well-known oncogene frequently up-regulated in different malignancies, that modulated glucose metabolism in various carcinoma [90, 91, 92]. Consequently, we guess that the combination of PKC $\delta$ with proteasome $\alpha$ 2 promoter may affect the activation of c-Myc, further modulated glucose metabolism to promote tumor progression.

PKC $\delta$ is thought to be an ambiguous role in carcinogenesis. Furthermore, numerous in vitro and in vivo studies have suggested that PKC $\delta$ have been linked to increased invasion, proliferation, drug resistance and genetic instability. UPS regulates degradation of proteins that is involved in cell proliferation, apoptosis, migration, and invasion. Disruption of UPS by mutations or modified expression of specific components within the cascade plays an important role in tumorigenesis. The notion supports a possibility of that PKC $\delta$ regulates the function of UPS by changing the composition of proteasome in the aggressiveness of a cancer. Human high-grade cancers very frequently display mutations in genes such as c-Myc, which lead to increased proliferation rate, metastasis, anti-apoptosis, and resistance to therapy. Altered PKC $\delta$ and imbalanced UPS may be a mediator of these pro-cancer events. Taken together, PKC $\delta$ regulates the function of UPS during breast cancer progression, and targeted therapies against PKC $\delta$ could be effective in the treatment cancers.

In summary, our hypothesis points to the role of PKC $\delta$ in the regulation of UPS in a subset of breast cancer patients with T2DM or hyperglycemia. The presence of PKC $\delta$ overexpression in breast cancer cases complicated with T2DM or hyperglycemia could worsen survival and contribute to cancer progression.