Breast cancer remains the most common cancer in women worldwide and the  second most common cancer among women in the U.S. The optimal treatment for  patients is based on the expression of the estrogen receptor α (ER), progesterone  receptor (PR) and HER2 as assessed by immunohistochemical methods. Patients with  tumors that express ER are treated with endocrine therapy including tamoxifen  or aromatase inhibitors administered in the adjuvant or neo-adjuvant setting.  Unfortunately a significant percentage of patients experience endocrine resistance  and disease recurrence. Such targeted therapies do not exist for patients with triple  negative breast cancer (TNBC), who are currently principally treated with cytotoxic  chemotherapy. Endocrine resistance in patients of ER+ subtype and the aggressive  nature of TNBC subtype continue to be significant roadblocks to successful therapeutic  management. Protein kinase C alpha (PKCα) is a serine-threonine kinase implicated  in numerous physiological and pathological processes, including breast cancer. PKCα participates in a number of oncogenic pathways, making it a very attractive target  for breast cancer therapy. Many of these pathways are functionally relevant in both  ER+ and TNBC patients. In addition, in ER+ patients, expression of PKCα is a promising  biomarker for poor response to endocrine therapy. This review will summarize the  current understanding of PKCα in both ER+ and TNBC subtypes. 

Protein Kinase C alpha ;Breast cancer ;Endocrine resistance ;Triple negative breast cancer.

Pham TND, Tonetti DA (2016) Protein Kinase C Alpha in Breast Cancer: A Focus on Endocrine Resistant and Triple Negative Breast Cancer. J Cancer Biol Res 4(1): 1076.

PKC: Protein Kinase C; ER: Estrogen Receptor; PR: Progesterone Receptor; HER2: Human Epidermal Growth Factor 2; EGFR: Epidermal Growth Factor Receptor; TNBC: Triple Negative Breast Cancer; TCGA: The Cancer Genome Atlas; CSC: Cancer Stem Cells; SERM: Selective Estrogen Receptor Modulator; SEM: Selective Estrogen Mimics; Sherpa: Selective Estrogen Receptor Partial Agonists

Breast cancer is the one of the most commonly diagnosed cancers among American women. Treatment success depends on multiple factors, including stage at diagnosis and the inherent features of the tumors. Over the last decade, major advances in microarray analysis have reproducibly established that breast cancers encompass several distinct disease entities, referred to as the intrinsic subtypes of breast cancer [1-4]. Among these, tumors of luminal subtype usually express estrogen receptor alpha (ER) and account for about 70% of breast cancer cases [5]. Basal-like breast cancer, which occurs in about 15-20% of breast cancer patients, is commonly known as triple negative breast cancer (TNBC) because the majority of cases lack expression of ER, progesterone receptor (PR), and HER2/neu amplification [6,7]. However, it is important to note that not all basal-like breast cancers are TNBC and not all TNBC are basal-like breast cancer. Basal-like phenotype is determined by microarray profiles whereas TNBC is based on immunohistochemical assays for ER, PR, and HER2 only. The reconciliation between molecular subtype and clinical subtype has been of great research interest, and several immunohistochemical markers have been proposed to define molecular subtypes. It is generally accepted that luminal tumors are ER+ and express many epithelial luminal genes including luminal cell keratin such as 8/18 [1,3,4]. However, the definition of basal breast cancer using immunohistochemical markers is more controversial. Several different panels have been proposed, the most often described is the expression of one or more high-molecular-weight/basal cytokeratins (CK5/6, CK14, and CK17) and/or EGFR in addition to the lack of ER, PR, and HER2 [6,8-10]. An insightful study by Prat et al compared clinical phenotype (immunohistochemitry-based) and molecular phenotype (microarray-based) and determined that approximately 78% of TNBC tumors are of the basal molecular subtype, and the rest are a mixture of HER2, luminal, and normallike [7]. Therefore, even though the two terms should not be used interchangeably, there is a remarkable overlap between TNBC and basal breast cancer.

Treatment options for ER+ and TNBC are different, but relapse following therapy resistance is a common clinical challenge. ER+ patients are routinely treated with endocrine therapies, which disrupt the ER-mediated signaling pathway either by blocking estrogen synthesis (aromatase inhibitors), promoting ER degradation (selective estrogen receptor downregulatorsSERDs), or competing with estrogen for ER (selective estrogen receptor modulator- SERMs). Tamoxifen is the most commonly prescribed SERM in the last 20 years both for the treatment of early and advanced breast cancer [11]. Unfortunately, a large proportion of women will relapse with acquired resistance [12]. TNBC patients, on the other hand, do not benefit from moleculartargeted therapy and often present an aggressive neoplasia with a strong association with distant recurrence and visceral metastases [13]. Patients of acquired resistance to hormonal therapies and of TNBC subtype present persistent challenges in the successful management of the disease. Ongoing efforts are focused on identifying and characterizing novel molecular targets that can serve as biomarkers for treatment response and/ or targets to overcome resistance.

Compared to healthy breast tissues, breast cancers display elevated expression and activity of protein kinase C (PKC) proteins [14-16]. Consisting of twelve isozymes, this family of serine-threonine protein kinases participates in a variety of cellular functions [17-20] (Figure 1).

Figure 1 Schematic representation of the primary structure of conventional, novel, and atypical PKCs. All PKCs consists of a regulatory domain and a catalytic domain connected together by a hinge region. The presence of the pseudosubstrate keeps the enzyme inactive and gets released once the enzyme translocates to and interacts with the membrane. The conventional isozymes are activated by diacylglycerol and Ca2+, novel by diacylglycerol alone, and atypical can be activated in the absence of both diacylglycerol and Ca2+

PKCα belongs to the classical subgroup that requires diacylglycerol, calcium, and phospholipid for its activity, and can support proliferation, differentiation, motility, and cell cycle progression in a tissue and cell-specific manner [18,21,22]. A functional role of PKCα has been implicated in a number of pathological conditions including cancers [18,21,23-25]. In this review, we will summarize the current understanding of PKCα in breast cancer, specifically for endocrine-resistant and TNBC.

Expression of PKCα in breast cancer

PKCα is ubiquitously expressed in various mammalian tissues. Expression of PKCα was found to be upregulated in some cancers (prostate, bladder, endometrial) and downregulated in others (colorectal, malignant renal cell carcinoma) [26-30]. The role of this isozyme in breast cancer is complex, since there are conflicting reports on PKCα expression levels in breast tumors compared to normal healthy breast tissues. Several immunohistochemical studies, including our own, found an overexpression of PKCα in breast tumors compared to nonmalignant breast tissues [31-33] whereas two independent research groups found the opposite [34,35]. Overall, these studies reported that expression of PKCα in breast tumors ranges from 12% to 78% [31-33,35,36] (Table 1).

Table 1: Immunohistochemistry studies on expression of PKCα using formalin-fixed, paraffin-embedded breast tissues.
Study	Number of tumors examined	Antibody used	Number of PKCα positive tumors
Lahn, M., et al. Oncology, 2004. PMID 15459489	17	anti-pS657 PKCα (Upstate Biotechnology)	2 (11.8%)
Kerfoot C., et al.  J Histochem Cytochem, 2004. PMID 14966210 1	46	anti-PKCα (H-7, Santa Cruz Biotechnology)	15 (32.6%)
Assender, J.W., et al. J Clin Pathol, 2007. PMID 17965220 2	70	anti?PKCα (IgG1 clone 4, Upstate Biotechnology)	34 (48.5%)
Lonne, G.K., et al., Mol Cancer, 2010. PMID 20398285 3	250	anti-PKCα (C-20, Santa Cruz Biotechnology)	101 (40.4%)
Tonetti, D.A., et al., Int J Breast Cancer, 2012. PMID 22500240 4	198	anti-PKCα (C-20, Santa Cruz Biotechnology)	154 (77.8%)
1, 3 Positivity based on staining intensity as follows:  0 lack of staining, 1 low staining, 2 moderate staining, and 3 strong staining.  Score ≥ 1 was considered positive. In 3, Cohort 1 and 2 were combined.  Samples not evaluated were not included in the calculation. 2 Positivity based on the median of the tumor epithelial HScore immunostaining.   HScore ≥ 110 was considered positive 4 Positivity based on frequency and intensity of staining as follows: Frequency: 0, 1=1-10%, 2=11-35%, 3=36-70%, 4≥70%; Intensity: 1-4.  Either intensity ≥ 1 and/or frequency ≥ 1 was considered positive.

In studies that report PKCα overexpression in breast tumors, expression of PKCα is significantly associated with adverse features in breast cancer patients, including shorter time to relapse and resistance to antiestrogen therapy [31,32,37]. We recently re-evaluated our own data using composite scores calculated from both staining intensity and frequency to identify the upper quartile of PKCα expression levels. Using this more stringent scoring criteria we estimated PKCα expression to be 20% (Table 2),

Table 2:  Summary of PKCα IHC findings from the study by Tonetti et al [33].
	Number of patients	Respective %
3-4 Intensity	53	27%
3-4 Frequency	85	43%
6-8 composite score	38	19%
Luminal A	9	15%
Luminal B	7	12%
HER2+	7	23%
TNBC	15	33%
Composite score is the sum of intensity and frequency of PKCα staining.  Total number of patients N = 198; including 58 luminal A, 59 luminal B, 31 HER2-enriched, and 45 TNBC.

representing a significant portion of patients whose tumors are predicted to express PKCα. The significance of the level of PKCα expression is as yet unclear.

The underlying mechanism for the induction of PKCα protein remains elusive. One study found that, compared to normal breast tissues, breast cancers do not display elevated levels of PKCα transcripts [38]. Among The Cancer Genome Atlas (TCGA) samples with high PKCα protein levels, we found that only 36% have correspondingly high levels of the transcript [39- 41]. Interestingly, when only tumors that are ER-negative were considered (N=174), almost 70% of patients with high PKCα protein expression also show elevated transcripts. This finding implies that the regulatory mechanism of PKCα protein is not straightforward and may vary according to different cellular contexts. In contrast, only 8% of patients display concomitant gain of copy number and high expression of PKCα [39-41], suggesting that gene amplification is not a major determinant of PKCα expression at least in the TCGA cohort. It is, however, important to note that since these microarray data present relative instead of absolute expression levels, it is not a simple task to reconcile mRNA and immunohistochemical data. Discrepancies between the available information on PKC expression at the protein and mRNA levels, measured by microarray, has been noted for PKCε [42].

Alternatively, elevated expression of PKCα protein may be a result of post-translational modifications that alter protein halflife. For example, syndecan-4, a transmembrane heparan proteoglycan, directly interacts with PKCα and enhances PKCα stability without affecting its mRNA stability or transcription level [43-46]. As a key adhesion molecule, syndecan-4 is ubiquitously expressed but often at low levels in normal tissues [47,48]. An immunohistochemical study found that syndecan-4 is expressed in about 70% of breast tumors [49], yet few studies have examined in depth the role of syndecan-4 in breast malignancy. TCGA breast cancer samples with high PKCα protein expression do not have higher mRNA level of syndecan-4 (SDC4) compared to samples with lower PKCα protein expression (TCGA, Nature 2012) [40,41]. A correlation analysis between PKCα and syndecan-4 at the protein level would be more informative. However, there are limited comprehensive cancer proteome databases that allowed us to perform this analysis. The Cancer Proteomics Atlas (TCPA) provides a user-friendly portal on protein expression data over a large number of cell lines and tumors including those from TCGA [50,51]. As of now, however, information on syndecan-4 is not yet publicly available. 

A recent study by Antal et al systematically characterized a number of cancer-associated PKC mutations in the TCGA dataset, and concluded that they are all loss of function and may act in a dominant-negative manner [52]. It appeared that only one PRKCA mutation was identified in breast (A444V), suggesting that mutations of this gene are not a common event in breast cancer. In fact, the authors noted that PKC isozymes are more often mutated in certain cancers (melanomas, colorectal, and lung squamous cell carcinoma) than in others including breast. In agreement with this, we found the frequency of PRKCA mutations among TCGA breast cancer samples to be only 1% [39-41]. The clinical significance of these mutations is unknown.

PKCα in endocrine-resistant, ER+ breast cancer

Expression of PKCα was first reported by our laboratory to confer tamoxifen resistance in an ER+ breast cancer model [53]. The prognostic value of PKCα in predicting endocrine resistance, response duration, breast cancer specific survival, and overall survival was thereafter demonstrated [31,32,37].

PKCα can transduce a multitude of signaling pathways, some of which have been implicated in the development of endocrine resistance. One of these pathways may involve alteration of the ER and ER-mediated signaling pathway. Expression of ERα and PKCα are inversely correlated in human breast cancer cell lines as well as clinical samples [14,31,53,54]. We and others have demonstrated that forced expression of PKCα in ER+ breast cancer cells results in either a reduction of ERα mRNA levels or down-regulation of ERα function [53-55], which contributes to the estrogen-independent and tamoxifen-resistant phenotype [53,56]. In clinical samples, up to 30% of patients with acquired resistance lose expression of ERα [57-59]. The exact mechanism by which PKCα down-regulates expression and function of ERα requires more comprehensive studies, but one proposed hypothesis is the ability of PKCα to inhibit c-Jun phosphorylation, a regulator of ERα transcriptional activity [54,60]. Elevated expression of EGFR/HER2 and its downstream pathways such as MAPK/ERK and AKT have been reported to accompany tamoxifen resistance [61-69]. Identified as a downstream effector of HER2, PKCα expression and activity were deemed critical for breast cancer cell invasion [70]. In turn, PKCα increases the level of recycled HER2 protein at the cell membrane [71]. Several lines of evidence clearly show that expression of HER2 can be amplified over the course of disease progression [58,72]. It is hence an intriguing hypothesis that expression of PKCα could be modulated over time and contributes to the emergence of therapy resistance. These findings suggest that co-targeting PKCα and EGFR/HER2 pathway may offer additional clinical benefits in patients with aggressive disease. For example, dual inhibition of PKCα and ERK resulted in apoptosis of tamoxifenresistant MCF-7 cells [73]; and dual inhibition of PKCα and Src had therapeutic implication in breast cancer metastasis [70]. Of significant interest, PKCα was recently shown to delay HER2 therapy resistance due to its ability to block Notch-1 signaling in BT474 and MDA-MB-453 cell lines [74]. The authors suggest PKCα is a biomarker for good prognosis in HER2+ patients, which is opposite to the well-established role of PKCα in ER+ patients. This discrepancy may primarily be attributed to the intrinsic differences between tumor subtypes, as the functions of PKCα are known to be very pleiotropic and cell type-dependent.

Over the years, therapy resistance has been strongly linked to the activity of breast cancer stem cells (CSCs) [75-78]. In that regard, Notch signaling has been implicated in the regulation of CSCs in both ductal carcinoma in situ and invasive carcinoma of the breast [79-81]. Indeed, inhibition of Notch-4 signaling was found to reverse the endocrine resistance phenotype [82,83]. PKCα was identified as a positive regulator of Notch-4 and its downstream effects in endocrine-resistant breast cancer [83]. It is reasonable to speculate that PKCα promotes resistance to endocrine therapy via promoting Notch-induced stem cell activity. In agreement with this, our laboratory showed that ectopic expression of PKCα in T47D breast cancer cells led to a significant increase in the number of CD44+/high/CD24-/low cells [84], indicative of stem cells [85].

A number of alternative signaling transducers and pathways have been suggested to be co-targeted for the clinical management of tamoxifen-resistant breast cancer [86-88]. The studies presented so far strongly point at PKCα as a promising therapeutic target for these patients. Our laboratory was the first to show that expression of PKCα in primary breast tumors is predictive of recurrence on tamoxifen treatment [37]. These PKCα-expressing tumors regressed upon treatment with both 17β-estradiol (E2) and the SERM raloxifene, although upon raloxifene withdrawal, tumor growth resumes [89]. Raloxifene has a favorable antiestrogenic profile in the uterus and has proven safety over 15 years of clinical use in postmenopausal osteoporosis and breast cancer chemoprevention. This observation led us to design and investigate the therapeutic efficacy of selective estrogen mimics (SEM) and selective estrogen receptor partial agonists (ShERPA), based on the benzothiophene scaffold of raloxifene [90,91]. In contrast to E2, SEM and ShERPA compounds induce tumor regression without uterine proliferation; a known unwanted side effect of both tamoxifen and E2 [90,91]. The anti-tumorigenic effect exerted by E2 and raloxifene is multifaceted, including repression of the pro-survival, anti-apoptotic Akt pathway, activation of the Fas/FasL apoptotic pathway, and extranuclear translocation of the ERα receptor [89,92]. We are currently investigating the molecular mechanism behind ERα translocation from the nucleus, and propose that detection of extranuclear ERα can be used to monitor therapeutic response in tamoxifenresistant, PKCα-expressing breast cancers [89]. It is noteworthy that SEM and ShERPA compounds did not promote in vivo growth of PKCα non-expressing T47D/neo tumors [90,91]. This finding highlights the excellent toxicity profile of our novel compounds as well as suggests a possible contribution of PKCα to the intertumor heterogeneity in terms of therapy response.

PKCα in TNBC

Compared to endocrine-resistant breast cancer, reports on the functions of PKCα in TNBC have been relatively few. Our laboratory recently reported TNBCs exhibit significantly higher levels of PKCα compared to tumors of other subtypes [33]. Interestingly, TNBCs display significantly higher levels of PKCα transcripts compared to other subtypes (Figure 2).

Figure 2 Patients of TNBC subtype (ER-, PR-, and HER-) have elevated expression of PKCα transcripts compared to non-TNBC patients. Metaanalysis was performed on four different datasets from OncomineTM.

In fact, several microarray studies reveal that PKCα expression ranges from 60% to 80% in TNBC compared to only 20-40% in non-TNBC patients, indicating a potential function of this kinase in TNBC (Oncomine™). Accordingly, inhibition of PKCα expression and activity in TNBC cells significantly reduces their cellular migration in vitro and metastasis in vivo [93, 94]. Several downstream effectors of PKCα have been identified, including CXCR4 and Rac1, expression of which can promote directional migration and metastatic potential of breast cancer cells [93-98]. It appears that PKCα modulates CXCR4 at both the transcript and post-transcript level [94]. Transcriptional regulation of CXCR4 is dynamic and involves a wide variety of signaling molecules, including NF-κB [99]. Further investigation on how PKCα interacts with these molecules will provide a better understanding of CXCR4 regulation by PKCα in breast cancer.

Similar to ER+ breast cancers, the involvement of PKCα in breast cancer stem cells has been demonstrated in TNBC [100,101]. It is thought-provoking that PKCα was shown to have a supportive role only in the breast cancer stem cell but not in the non-stem cell population; therefore, targeting PKCα was more efficient at killing the former [100,101]. In both studies, PKCα expression and activity were crucial for the survival and tumor-initiating capacity of TNBC compared to non-TNBC cells, implying that this particular subtype may rely more heavily on the stem cell population for tumor initiation and maintenance. These results highlight the well-known inter- as well as intratumor heterogeneity of breast cancer. As a result, co-targeting cancer stem and non-stem cell population will likely offer superior therapeutic effects for the treatment of the disease.

In addition, the study by Tam et al identified PDGFR as the principle pathway downstream of PKCα [101]. This finding, in companion with the known crosstalk between PKCα and EGFR/ HER2 in ER+ breast cancers underscores the functional diversity of PKCα in various cell types. Notably, the FOXC2 transcription factor was recently reported to be an upstream regulator of PDGFRβ in TNBC [102]. These independent reports are suggestive of a functional link between PKCα and FOXC2 in TNBC and TNBC stem cells. Our data demonstrate that in both TNBC and ER+ , endocrine resistant cell lines, PKCα is a positive regulator of FOXC2, and this regulation likely involves transcriptional modulation of FOXC2, leading to its overexpression [103]. Indeed, several independent studies have showed that TNBC tumors display elevated expression of FOXC2 compared to nonTNBC tumors [102-104]. Our finding therefore provides a novel underlying mechanism for the induction of FOXC2 expression in TNBC tumors. Lastly, co-expression of PKCα and FOXC2 is significantly associated with poorer relapse-free survival in breast cancer patients of ER-negative, basal intrinsic subtype, as determined by Kaplan-Meier analysis (Figure 3A)

Figure 3 Patients whose tumors co-express high levels of PKCα and FOXC2 experience significant shorter relapse free survival compared to those whose tumors do not. A. Patients of ER-negative, basal subtype, N=339.

 [105]. The predictive value of PKCα and FOXC2 co-expression, on the other hand, was not as obvious in ER+ patients (Figure 3B)

Figure 3 Patients whose tumors co-express high levels of PKCα and FOXC2 experience significant shorter relapse free survival compared to those whose tumors do not.B. Patients of ER-positive subtype, N=1802.

[105]. When patients were stratified based on treatment received (endocrine versus chemotherapy), the co-expression had no significant value at predicting their relapse free survival.

Similar to what was observed in endocrine-resistant, ER+ breast cancer, expression of Notch receptors, such as Notch-4, and their ligands are elevated in TNBC [106-109]. Inhibition of Notch signaling reduces colony formation and xenograft growth of TNBC cells [107,110]. It will be of great interest to determine whether there is a functional relationship between PKCα and Notch, as observed in ER+ breast cancer cells, also holds true in the TNBC subtype. Therefore, a growing body of evidence highlights PKCα as a therapeutic target in TNBC.

PKCα as a common therapeutic target for ER+, endocrine resistant and TNBC

Hormone resistance remains a significant roadblock to the successful treatment of luminal tumors. One possible mechanism of resistance is the existence of the CSC population [75-78]. Originally identified as cells having CD24-/low/CD44+/ high marker profile, CSCs have the ability to initiate tumors in immunocompromised or syngeneic mice, self-renewal capacity measured by tumor formation in secondary mice, and the capacity to differentiate into the non-self-renewing cells, which constitute the tumor bulk [85]. They do not only possess higher inherent resistance to therapies, but also reportedly thrive with these treatments [75,82,85]. A subpopulation of ER- /PR- / CD44+ /cytokeratin (CK) 5+ cells that share the properties of CSCs has been identified in ER+ /PR+ breast cancer xenografts [111]. Treatment with tamoxifen or fulvestrant led to selective enrichment of these cells, whereas the population of ER+ /PR+ cells was decreased [112]. Importantly, Haughian et al revealed that this subpopulation of ER- /PR- /CD44+ /CK5+ cells in luminal tumors displays a gene expression profile similar to that of TNBC/ basal tumors, and therefore was named as luminobasal [113]. Intriguingly, ER- /PR- /CD44+ /CK5+ cells also depend on Notch signaling for survival and proliferation. These findings suggest that different breast cancer subtypes may rely, albeit to different extents, on similar pathways for their oncogenic behaviors. Here we have described evidence to suggest that PKCα is a promising target for both ER+ , endocrine-resistant and basal/TNBC. With the ability to crosstalk to a number of oncogenic pathways, PKCα presents itself as a novel therapeutic target that is functionally important in multiple cell lineages and can be exploited for the effective management of breast cancer. A simplified summary of this crosstalk is depicted in Figure 4.

Figure 4 Crosstalk between PKCα and several key signaling pathways. References are indicated in brackets.

Several approaches are used to target PKC isozymes therapeutically. These inhibitors either compete for PKCα substrates, ATP, or interfere with the binding of activated PKCα to the membrane [23,114-116]. In general, these compounds do not have selective specificity for PKCα due to a high degree of structural conservation with other PKC isozymes. Past clinical trials with PKC modulators, either by themselves or with other anti-cancer modalities were disappointing due to their lack of therapeutic efficacy [23,114-116]. Despite promising preliminary results with the antisense oligonucleotide Aprinocarsen (ISI 3521, LY900003) specifically targeting human PKCα mRNA, no clinical benefit was observed in a small phase II with metastatic breast cancer patients [117,118]. This outcome is multifaceted, and could in part be due to the lack of prior screening for PKCα expression in patients receiving treatment. Further investigation for aprinocarsen was discontinued after a few more unsuccessful trials [119-124].

A relatively more recent attempt to develop selective PKC inhibitors focuses on disrupting the interactions between PKCs and anchoring proteins, which play a critical role in positioning activated PKCs to various cellular compartments, including the cell membrane. Many of these interactions map to the C2, V2, V3 and V5 regions [125] (Figure 1). Pioneer work done by MochlyRosen’s lab demonstrates that several of them are isozymespecific and, therefore, can be targeted to achieve selective inhibition [116]. αV5-3 was designed from the V5 region of PKCα based on this rational approach [94]. In vivo administration of this agent completely abrogated metastasis of breast cancer cells to lung [94]. In addition, no signs of toxicity were observed in the animals, making αV5-3 a promising agent for the management of mammary tumor metastasis. Mechanistic studies found that reduction of MMP9 and NF-κB activity was a downstream effect of PKCα inhibition by αV5-3 and contributed to the peptide’s anti-metastasis property [94]. Of interest, another research group found that curcumin and decursin can also inhibit PKCα translocation, and reduce MMP9 and NF-κB activity in breast cancer [126,127]. Curcumin, which is extracted from the plant Curcuma longa, is recognized for its chemopreventive and antitumoral activities against various cancers, including breast [128,129]. Similarly, studies that demonstrate the efficacy of decursin, a pyranocoumarin, have been reported for both TNBC [130,131] and ER+ breast cancer [131]. Whereas curcumin is able to inhibit PKC activation by competing with both phosphatidylserine and calcium [132,133], the exact mechanism whereby decursin inhibits PKCα translocation remains to be studied in depth.

Of note, several miRNAs were discovered that target PKCα transcripts [93,134,135]. Among these, decreased miR-200b expression in TNBC was observed, likely contributing to elevated PKCα expression. Ectopic expression of miR-200b drastically reduced TNBC cell migration and inhibited tumor metastasis in an orthotopic mouse mammary xenograft tumor model [93]. Similarly, reduced miR-200b expression was demonstrated to contribute to endocrine resistance in breast cancer cells [136]. Over expression of miR-200b sensitized resistant cells to growth inhibition by tamoxifen [136]. Recent advancement in the field of in vivo miRNA delivery will allow us to therapeutically evaluate and compare the magnitude of the effect mediated by miR-200b in ER+ versus TNBC patients.

Clearly, there is a need for a more stringent and clinically relevant evaluation of the significance and prevalence of PKCα expression in breast cancer patients. Regardless, PKCα is a promising therapeutic target for the treatment of various breast cancer subtypes. We are evaluating the expression of PKCα as a biomarker for endocrine therapy response in ER+ breast cancer patients in companion with novel SEM and ShERPA compounds that could offer therapeutic benefit to tamoxifen resistant patients. Specific targeting of PKCα in a cell type- and tissuespecific manner remains a challenge as the high conservation of domain structures among PKC members prevent selective inhibitors of PKCα to be effectively transitioned to the clinic.

1. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000; 406: 747-752.

2. Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol. 2009; 27: 1160-1167.

3. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98: 10869-10874.

4. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003; 100: 8418-8423.

5. Dunnwald LK, Rossing MA, Li CI. Hormone receptor status, tumor characteristics, and prognosis: a prospective cohort of breast cancer patients. Breast Cancer Res. 2007; 9: R6.

6. Badve S, Dabbs DJ, Schnitt SJ, Baehner FL, Decker T, Eusebi V, et al. Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod Pathol. 2011; 24: 157-167.

7. Prat A, Adamo B, Cheang MC, Anders CK, Carey LA, Perou CM. Molecular characterization of basal-like and non-basal-like triplenegative breast cancer. Oncologist. 2013; 18: 123-133.

8. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004; 10: 5367-5374.

9. Cheang MC, Voduc D, Bajdik C, Leung S, McKinney S, Chia SK, et al. Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 2008; 14: 1368-1376.

10. Bertucci F, Finetti P, Birnbaum D. Basal breast cancer: a complex and deadly molecular subtype. Curr Mol Med. 2012; 12: 96-110.

11. Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet. 1998; 351: 1451-1467. 12.Howell A, Wardley AM. Overview of the impact of conventional systemic therapies on breast cancer. Endocr Relat Cancer. 2005; 12: 9-16.

13. Anders CK, Carey LA. Biology, metastatic patterns, and treatment of patients with triple-negative breast cancer. Clin Breast Cancer. 2009; 9: 73-81.

14. Borner C, Wyss R, Regazzi R, Eppenberger U, Fabbro D. Immunological quantitation of phospholipid/Ca2+-dependent protein kinase of human mammary carcinoma cells: inverse relationship to estrogen receptors. Int J Cancer. 1987; 40: 344-348.

15. Gordge PC, Hulme MJ, Clegg RA, Miller WR. Elevation of protein kinase A and protein kinase C activities in malignant as compared with normal human breast tissue. Eur J Cancer. 1996; 32A: 2120-2126.

16. O’Brian C, Vogel VG, Singletary SE, Ward NE. Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res. 1989; 49: 3215-3217.

17. Caino MC, von Burstin VA, Lopez-Haber C, Kazanietz MG. Differential regulation of gene expression by protein kinase C isozymes as determined by genome-wide expression analysis. J Biol Chem. 2011; 286: 11254-11264.

18. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol. 2000; 279: L429-438.

19. Gutcher I, Webb PR, Anderson NG. The isoform-specific regulation of apoptosis by protein kinase C. Cell Mol Life Sci. 2003; 60: 1061-1070.

20. Hata A, Akita Y, Suzuki K, Ohno S. Functional divergence of protein kinase C (PKC) family members. PKC gamma differs from PKC alpha and -beta II and nPKC epsilon in its competence to mediate-12-Otetradecanoyl phorbol 13-acetate (TPA)-responsie transcriptional activation through a TPA-response element. J Biol Chem. 1993; 268: 9122-9129.

21. Nakashima S. Protein kinase C alpha (PKC alpha): regulation and biological function. J Biochem. 2002; 132: 669-675.

22. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci. 2004; 117: 131- 132.

23. Konopatskaya O, Poole AW. Protein kinase Calpha: disease regulator and therapeutic target. Trends Pharmacol Sci. 2010; 31: 8-14.

24. Koivunen J, Aaltonen V, Peltonen J. Protein kinase C (PKC) family in cancer progression. Cancer Lett. 2006; 235: 1-10.

25. Michie AM, Nakagawa R. The link between PKCalpha regulation and cellular transformation. Immunol Lett. 2005; 96: 155-162.

26. Koren R, Ben Meir D, Langzam L, Dekel Y, Konichezky M, Baniel J, et al. Expression of protein kinase C isoenzymes in benign hyperplasia and carcinoma of prostate. Oncol Rep. 2004; 11: 321-326.

27. Fournier DB, Chisamore M, Lurain JR, Rademaker AW, Jordan VC, Tonetti DA. Protein kinase C alpha expression is inversely related to  ER status in endometrial carcinoma: possible role in AP-1-mediated proliferation of ER-negative endometrial cancer. Gynecol Oncol. 2001; 81: 366-372.

28. Suga K, Sugimoto I, Ito H, Hashimoto E. Down-regulation of protein kinase C-alpha detected in human colorectal cancer. Biochem Mol Biol Int. 1998; 44: 523-528.

29. von Brandenstein M, Pandarakalam JJ, Kroon L, Loeser H, Herden J, Braun G, et al. MicroRNA 15a, inversely correlated to PKCα, is a potential marker to differentiate between benign and malignant renal tumors in biopsy and urine samples. Am J Pathol. 2012; 180: 1787- 1797.

30. Varga A, Czifra G, Tállai B, Németh T, Kovács I, Kovács L, et al. Tumor grade-dependent alterations in the protein kinase C isoform pattern in urinary bladder carcinomas. Eur Urol. 2004; 46: 462-465.

31. Assender JW, Gee JM, Lewis I, Ellis IO, Robertson JF, Nicholson RI. Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer. J Clin Pathol. 2007; 60: 1216- 1221.

32. Lønne GK, Cornmark L, Zahirovic IO, Landberg G, Jirström K, Larsson C. PKCalpha expression is a marker for breast cancer aggressiveness. Mol Cancer. 2010; 9: 76.

33. Tonetti DA, Gao W, Escarzaga D, Walters K, Szafran A, Coon JS. PKCα and ERβ Are Associated with Triple-Negative Breast Cancers in African American and Caucasian Patients. Int J Breast Cancer. 2012; 2012: 740353.

34. Ainsworth PD, Winstanley JH, Pearson JM, Bishop HM, Garrod DR. Protein kinase C alpha expression in normal breast, ductal carcinoma in situ and invasive ductal carcinoma. Eur J Cancer. 2004; 40: 2269- 2273.

35.  Kerfoot C, Huang W, Rotenberg SA. Immunohistochemical analysis of advanced human breast carcinomas reveals downregulation of protein kinase C alpha. J Histochem Cytochem. 2004; 52: 419-422.

36. Lahn M, Köhler G, Sundell K, Su C, Li S, Paterson BM, et al. Protein kinase C alpha expression in breast and ovarian cancer. Oncology. 2004; 67: 1-10.

37. Tonetti DA, Morrow M, Kidwai N, Gupta A, Badve S. Elevated protein kinase C alpha expression may be predictive of tamoxifen treatment failure. Br J Cancer. 2003; 88: 1400-1402.

38. Awadelkarim KD, Callens C, Rossé C, Susini A, Vacher S, Rouleau E, et al. Quantification of PKC family genes in sporadic breast cancer by qRT-PCR: evidence that PKCι/λ overexpression is an independent prognostic factor. Int J Cancer. 2012; 131: 2852-2862.

39. Ciriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al. Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer. Cell. 2015; 163: 506-519.

40. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012; 2: 401- 404.

41. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013; 6: pl1.

42. Garg R, Benedetti LG, Abera MB, Wang H, Abba M, Kazanietz MG. Protein kinase C and cancer: what we know and what we do not. Oncogene. 2014; 33: 5225-5237.

43. Keum E, Kim Y, Kim J, Kwon S, Lim Y, Han I, et al. Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha. Biochem J. 2004; 378: 1007-1014.

44. Oh ES, Woods A, Couchman JR. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C. J Biol Chem. 1997; 272: 8133-8136.

45. Oh ES, Woods A, Couchman JR. Multimerization of the cytoplasmic domain of syndecan-4 is required for its ability to activate protein kinase C. J Biol Chem. 1997; 272: 11805-11811.

46. Oh ES, Woods A, Lim ST, Theibert AW, Couchman JR. Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity. J Biol Chem. 1998; 273: 10624-10629. 47.Couchman JR. Syndecans: proteoglycan regulators of cell-surface microdomains? Nat Rev Mol Cell Biol. 2003; 4: 926-937.

48. Morgan MR, Humphries MJ, Bass MD. Synergistic control of cell adhesion by integrins and syndecans. Nat Rev Mol Cell Biol. 2007; 8: 957-969.

49. Lendorf ME, Manon-Jensen T, Kronqvist P, Multhaupt HA, Couchman JR. Syndecan-1 and syndecan-4 are independent indicators in breast carcinoma. J Histochem Cytochem. 2011; 59: 615-629.

50. Li J, Lu Y, Akbani R, Ju Z, Roebuck PL, Liu W, et al. TCPA: a resource for cancer functional proteomics data. Nat Methods. 2013; 10: 1046- 1047.

51. Akbani R, Ng PK, Werner HM, Shahmoradgoli M, Zhang F, Ju Z, et al. A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nat Commun. 2014; 5: 3887.

52. Antal CE, Hudson AM, Kang E, Zanca C, Wirth C, Stephenson NL, et al. Cancer-associated protein kinase C mutations reveal kinase’s role as tumor suppressor. Cell. 2015; 160: 489-502.

53. Tonetti DA, Chisamore MJ, Grdina W, Schurz H, Jordan VC. Stable transfection of protein kinase C alpha cDNA in hormone-dependent breast cancer cell lines. Br J Cancer. 2000; 83: 782-791.

54. Kim S, Lee J, Lee SK, Bae SY, Kim J, Kim M, et al. Protein kinase C-α downregulates estrogen receptor-α by suppressing c-Jun phosphorylation in estrogen receptor-positive breast cancer cells. Oncol Rep. 2014; 31: 1423-1428.

55. Ways DK, Kukoly CA, deVente J, Hooker JL, Bryant WO, Posekany KJ, et al. MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest. 1995; 95: 1906-1915.

56. Chisamore MJ, Ahmed Y, Bentrem DJ, Jordan VC, Tonetti DA. Novel antitumor effect of estradiol in athymic mice injected with a T47D breast cancer cell line overexpressing protein kinase Calpha. Clin Cancer Res. 2001; 7: 3156-3165.

57. Johnston SR, Saccani-Jotti G, Smith IE, Salter J, Newby J, Coppen M, et al. Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res. 1995; 55: 3331-3338.

58. Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, Allred DC, et al. Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. J Clin Oncol. 2005; 23: 2469-2476.

59. Dowsett M. Overexpression of HER-2 as a resistance mechanism to hormonal therapy for breast cancer. Endocr Relat Cancer. 2001; 8: 191-195.

60. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, et al. Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol. 2000; 74: 311-317.

61. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S,  Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem. 2001; 276: 9817-9824.

62. Ciocca DR, Elledge R. Molecular markers for predicting response to tamoxifen in breast cancer patients. Endocrine. 2000; 13: 1-10.

63. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, et al. Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology. 2003; 144: 1032- 1044.

64. Mass R. The role of HER-2 expression in predicting response to therapy in breast cancer. Semin Oncol. 2000; 27: 46-52.

65. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, et al. Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst. 2003; 95: 353-361.

66. Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, et al. Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res Treat. 1992; 24: 85-95.

67. Liu Y, el-Ashry D, Chen D, Ding IY, Kern FG. MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogendependence and tamoxifen-sensitivity in vivo. Breast Cancer Res Treat. 1995; 34: 97-117.

68. Pietras RJ, Arboleda J, Reese DM, Wongvipat N, Pegram MD, Ramos L, et al. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene. 1995; 10: 2435-2446.

69. Fan M, Yan PS, Hartman-Frey C, Chen L, Paik H, Oyer SL, et al. Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant. Cancer Res. 2006; 66: 11954-11966.

70. Tan M, Li P, Sun M, Yin G, Yu D. Upregulation and activation of PKC alpha by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKC alpha and Src inhibitors. Oncogene. 2006; 25: 3286-3295.

71. Magnifico A, Albano L, Campaner S, Campiglio M, Pilotti S, Ménard S, et al. Protein kinase Calpha determines HER2 fate in breast carcinoma cells with HER2 protein overexpression without gene amplification. Cancer Res. 2007; 67: 5308-5317.

72. Massarweh S, Osborne CK, Creighton CJ, Qin L, Tsimelzon A, Huang S, et al. Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function. Cancer Res. 2008; 68: 826-833.

73. Li Z, Wang N, Fang J, Huang J, Tian F, Li C, et al. Role of PKC-ERK signaling in tamoxifen-induced apoptosis and tamoxifen resistance in human breast cancer cells. Oncol Rep. 2012; 27: 1879-1886.

74. Pandya K, Wyatt D, Gallagher B, Shah D, Baker A, Bloodworth J, et al. PKCα Attenuates Jagged-1-Mediated Notch Signaling in ErbB2-Positive Breast Cancer to Reverse Trastuzumab Resistance. Clin Cancer Res. 2016; 22: 175-186.

75. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci U S A. 2009; 106: 13820-13825.

76. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008; 100: 672-679. 77.Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005; 5: 275-284.

78. O’Brien CS, Farnie G, Howell SJ, Clarke RB. Breast cancer stem cells and their role in resistance to endocrine therapy. Horm Cancer. 2011; 2: 91-103.

79. Al Saleh S, Sharaf LH, Luqmani YA. Signalling pathways involved in endocrine resistance in breast cancer and associations with epithelial to mesenchymal transition (Review). Int J Oncol. 2011; 38: 1197-1217.

80. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res. 2004; 6: R605- 615. 81.Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res. 2006; 66: 1517-1525.

82. Simões BM, O’Brien CS, Eyre R, Silva A, Yu L, Sarmiento-Castro A, et al. Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity. Cell Rep. 2015; 12: 1968-1977.

83. Yun J, Pannuti A, Espinoza I, Zhu H, Hicks C, Zhu X, et al. Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells. Oncogenesis. 2013; 2: e60.

84. White B. Protein Kinase C Alpha Destabilizes Adherens Junctions via p120-Catenin in Human Breast Cancer [dissertation]: University of Illinois at Chicago; 2012. 85.Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003; 100: 3983-3988.

86. Milani A, Geuna E, Mittica G, Valabrega G. Overcoming endocrine resistance in metastatic breast cancer: Current evidence and future directions. World J Clin Oncol. 2014; 5: 990-1001.

87. Zhao M, Ramaswamy B. Mechanisms and therapeutic advances in the management of endocrine-resistant breast cancer. World J Clin Oncol. 2014; 5: 248-262. 88.Ziauddin MF, Hua D, Tang SC. Emerging strategies to overcome resistance to endocrine therapy for breast cancer. Cancer Metastasis Rev. 2014; 33: 791-807.

89. Perez White B, Molloy ME, Zhao H, Zhang Y, Tonetti DA. Extranuclear ERα is associated with regression of T47D PKCα -overexpressing, tamoxifen-resistant breast cancer. Mol Cancer. 2013; 12: 34.

90. Molloy ME, White BE, Gherezghiher T, Michalsen BT, Xiong R, Patel H, et al. Novel selective estrogen mimics for the treatment of tamoxifenresistant breast cancer. Mol Cancer Ther. 2014; 13: 2515-2526.

91. Xiong R, Patel HK, Gutgesell LM, Zhao J, Delgado-Rivera L, Pham TN, et al. Selective Human Estrogen Receptor Partial Agonists (ShERPAs) for Tamoxifen-Resistant Breast Cancer. J Med Chem. 2016; 59: 219-237.

92. Zhang Y, Zhao H, Asztalos S, Chisamore M, Sitabkhan Y, et al. Estradiolinduced regression in T47D:A18/PKCalpha tumors requires the estrogen receptor and interaction with the extracellular matrix. Mol Cancer Res. 2009; 7: 498-510.

93. Humphries B, Wang Z, Oom AL, Fisher T, Tan D, Cui Y, et al. MicroRNA200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis. Carcinogenesis. 2014; 35: 2254-2263.

94. Kim J, Thorne SH, Sun L, Huang B, Mochly-Rosen D. Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model. Oncogene. 2011; 30: 323-333.

95. Harvey JR, Mellor P, Eldaly H, Lennard TW, Kirby JA, Ali S. Inhibition of CXCR4-mediated breast cancer metastasis: a potential role for heparinoids? Clin Cancer Res. 2007; 13: 1562-1570.

96. Mimeault M, Batra SK. Functions of tumorigenic and migrating cancer progenitor cells in cancer progression and metastasis and their therapeutic implications. Cancer Metastasis Rev. 2007; 26: 203-214.

97. Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, et al. CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res. 2004; 64: 8604-8612.

98. Wertheimer E, Gutierrez-Uzquiza A, Rosemblit C, Lopez-Haber C, Sosa MS, Kazanietz MG. Rac signaling in breast cancer: a tale of GEFs and GAPs. Cell Signal. 2012; 24: 353-362.

99. Busillo JM, Benovic JL. Regulation of CXCR4 signaling. Biochim Biophys Acta. 2007; 1768: 952-963.

100. Hsu YH, Yao J, Chan LC, Wu TJ, Hsu JL, Fang YF, et al. Definition of PKC-α, CDK6, and MET as therapeutic targets in triple-negative breast cancer. Cancer Res. 2014; 74: 4822-4835.

101. Tam WL, Lu H, Buikhuisen J, Soh BS, Lim E, Reinhardt F, et al. Protein kinase C α is a central signaling node and therapeutic target for breast cancer stem cells. Cancer Cell. 2013; 24: 347-364.

102. Hollier BG, Tinnirello AA, Werden SJ, Evans KW, Taube JH, Sarkar TR, et al. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Res. 2013; 73: 1981- 1992.

103. Pham TW, BPW; Tonetti, DA, editor. Protein kinase C alpha (PKCa) is a novel regulator of FOXC2 and p120-catenin in triple negative and endocrine resistant breast cancer Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015; Philadelphia, PA: Cancer Res.

104. Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, et al. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci U S A. 2007; 104: 10069-10074.

105. Gy?rffy B, Surowiak P, Budczies J, Lánczky A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One. 2013; 8: e82241. 106. Lee CW, Simin K, Liu Q, Plescia J, Guha M, Khan A, et al. A functional Notch-survivin gene signature in basal breast cancer. Breast Cancer Res. 2008; 10: 97.

107. Nagamatsu I, Onishi H, Matsushita S, Kubo M, Kai M, Imaizumi A, et al. NOTCH4 is a potential therapeutic target for triple-negative breast cancer. Anticancer Res. 2014; 34: 69-80.

108. Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, et al. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res. 2005; 65: 8530-8537.

109. Reedijk M, Pinnaduwage D, Dickson BC, Mulligan AM, Zhang H, Bull SB, et al. JAG1 expression is associated with a basal phenotype and recurrence in lymph node-negative breast cancer. Breast Cancer Res Treat. 2008; 111: 439-448.

110. Zhang S, Chung WC, Miele L, Xu K. Targeting Met and Notch in the Lfng-deficient, Met-amplified triple-negative breast cancer. Cancer Biol Ther. 2014; 15: 633-642.

111. Horwitz KB, Dye WW, Harrell JC, Kabos P, Sartorius CA. Rare steroid receptor-negative basal-like tumorigenic cells in luminal subtype human breast cancer xenografts. Proc Natl Acad Sci U S A. 2008; 105: 5774-5779.

112. Kabos P, Haughian JM, Wang X, Dye WW, Finlayson C, Elias A, et al. Cytokeratin 5 positive cells represent a steroid receptor negative and therapy resistant subpopulation in luminal breast cancers. Breast Cancer Res Treat. 2011; 128: 45-55.

113. Haughian JM, Pinto MP, Harrell JC, Bliesner BS, Joensuu KM, Dye WW, et al. Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch. Proc Natl Acad Sci U S A. 2012; 109: 2742-2747. 114. Hofmann J. Protein kinase C isozymes as potential targets for anticancer therapy. Curr Cancer Drug Targets. 2004; 4: 125-146.

115. Dowling CM, Kiely PA. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling. Cancers (Basel). 2015; 7: 1271-1291.

116.  Mochly-Rosen D, Das K, Grimes KV. Protein kinase C, an elusive therapeutic target? Nat Rev Drug Discov. 2012; 11: 937-957.

117. Li K, Zhang J. ISIS-3521. Isis Pharmaceuticals. Curr Opin Investig Drugs. 2001; 2: 1454-1461. 118. Mackay HJ, Twelves CJ. Protein kinase C: a target for anticancer drugs? Endocr Relat Cancer. 2003; 10: 389-396.

119. Cripps MC, Figueredo AT, Oza AM, Taylor MJ, Fields AL, Holmlund JT, et al. Phase II randomized study of ISIS 3521 and ISIS 5132 in patients with locally advanced or metastatic colorectal cancer: a National Cancer Institute of Canada clinical trials group study. Clin Cancer Res. 2002; 8: 2188-2192.

120. Tolcher AW, Reyno L, Venner PM, Ernst SD, Moore M, Geary RS, et al. A randomized phase II and pharmacokinetic study of the antisense oligonucleotides ISIS 3521 and ISIS 5132 in patients with hormonerefractory prostate cancer. Clin Cancer Res. 2002; 8: 2530-2535. 121. Marshall JL, Eisenberg SG, Johnson MD, Hanfelt J, Dorr FA, El-Ashry D, et al. A phase II trial of ISIS 3521 in patients with metastatic colorectal cancer. Clin Colorectal Cancer. 2004; 4: 268-274.

122. Advani R, Peethambaram P, Lum BL, Fisher GA, Hartmann L, Long HJ, et al. A Phase II trial of aprinocarsen, an antisense oligonucleotide inhibitor of protein kinase C alpha, administered as a 21-day infusion to patients with advanced ovarian carcinoma. Cancer. 2004; 100: 321-326.

123. Rao S, Watkins D, Cunningham D, Dunlop D, Johnson P, Selby P, et al. Phase II study of ISIS 352, an antisense oligodeoxynucleotide to protein kinase C alpha, in patients with previously treated low-grade non-Hodgkin’s lymphoma. Ann Oncol. 2004; 15: 1413-1418.

124. Paz-Ares L, Douillard JY, Koralewski P, Manegold C, Smit EF, Reyes JM, et al. Phase III study of gemcitabine and cisplatin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleotide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol. 2006; 24: 1428-1434.

125. Kheifets V, Mochly-Rosen D. Insight into intra- and inter-molecular interactions of PKC: design of specific modulators of kinase function. Pharmacol Res. 2007; 55: 467-476.

126. Kim JM, Noh EM, Kim MS, Hwang JK, Hwang HY, Ryu DG, et al. Decursin prevents TPA-induced invasion through suppression of PKCα/p38/NF-κB-dependent MMP-9 expression in MCF-7 human breast carcinoma cells. Int J Oncol. 2014; 44: 1607-1613.

127. Kim JM, Noh EM, Kwon KB, Kim JS, You YO, Hwang JK, et al. Curcumin suppresses the TPA-induced invasion through inhibition of PKCα- dependent MMP-expression in MCF-7 human breast cancer cells. Phytomedicine. 2012; 19: 1085-1092.

128. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple biological activities of curcumin: a short review. Life Sci. 2006; 78: 2081-2087.

129. Liu D, Chen Z. The effect of curcumin on breast cancer cells. J Breast  Cancer. 2013; 16: 133-137.

130. Kim JH, Jung JH, Kim SH, Jeong SJ. Decursin exerts anti-cancer activity in MDA-MB-231 breast cancer cells via inhibition of the Pin1 activity and enhancement of the Pin1/p53 association. Phytother Res. 2014; 28: 238-244.

131. Jiang C, Guo J, Wang Z, Xiao B, Lee HJ, Lee EO, et al. Decursin and decursinol angelate inhibit estrogen-stimulated and estrogenindependent growth and survival of breast cancer cells. Breast Cancer Res. 2007; 9: R77.

132. Liu JY, Lin SJ, Lin JK. Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells. Carcinogenesis. 1993; 14: 857-861.

133. Mahmmoud YA. Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions. Br J Pharmacol. 2007; 150: 200-208.

134. Wang C, Wang X, Liang H, Wang T, Yan X, Cao M, et al. miR-203 inhibits cell proliferation and migration of lung cancer cells by targeting PKCα. PLoS One. 2013; 8: e73985.

135. Martin EC, Elliott S, Rhodes LV, Antoon JW, Fewell C, Zhu Y, et al. Preferential star strand biogenesis of pre-miR-24-2 targets PKCalpha and suppresses cell survival in MCF-7 breast cancer cells. Mol Carcinog. 2014; 53: 38-48.

136. Manavalan TT, Teng Y, Litchfield LM, Muluhngwi P, Al-Rayyan N, et al. Reduced expression of miR-200 family members contributes to antiestrogen resistance in LY2 human breast cancer cells. PLoS One. 2013; 8: e62334.