YAP activation and Hippo signaling suppression by PKC eta promote triple-negative breast cancer metastasis

doi:10.21203/rs.3.rs-3238222/v1

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YAP activation and Hippo signaling suppression by PKC eta promote triple-negative breast cancer metastasis

https://doi.org/10.21203/rs.3.rs-3238222/v1

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Breast cancer is the leading cause of cancer-related deaths in women, with metastasis being the primary reason for mortality. Patients with triple-negative breast cancer (TNBC) show an increased risk of metastatic dissemination. Protein kinase C eta (PKCη), an anti-apoptotic kinase of the novel PKC subfamily, is associated with poor prognosis in breast cancer patients. Here, we demonstrate that PKCη promotes metastasis in TNBC cells and show that this is mediated by the PKCƞ-YAP signaling axis. Knockout of PKCη (PKCη^KO) in the TNBC cells, 4T1 and MDA-MB-231, markedly inhibited their invasion and migration capability. Furthermore, orthotopic xenografts of the latter PKCη^KO cells in NSG mice reduced tumor growth and lung metastasis compared to PKCη-intact tumors. Mechanistically, we show that PKCη regulates epithelial-to-mesenchymal transition (EMT), as knockout of PKCη in TNBC cell lines increased expression of the EMT markers E-cadherin, EpCAM, and slug, and decreased expression of vimentin, ZEB1. Further profiling of the Hippo-YAP axis showed that PKCη is a negative regulator of the Hippo pathway that leads to YAP stabilization and its phosphorylation at Ser128, which allows YAP to translocate to the nucleus and contribute to metastasis of TNBC cells. We further show that PKCη directly interacts with YAP in silico and TNBC cells. Lastly, we demonstrate that treatment of TNBC cells with uPEP2, a recently discovered PKCη kinase inhibitory peptide (encoded by a uORF upstream of PKCη coding sequence), activates the Hippo pathway and YAP degradation. In summary, our results highlight the impact of PKCη in TNBC metastasis and offer a novel avenue for therapeutic intervention in this aggressive and fatal disease.

PKCη

uORF-encoded peptide

breast cancer

EMT

Metastasis

YAP

Hippo pathway

Breast cancer (BC) is the leading cause of cancer-related deaths in women [1]. Metastasis is the major reason for BC patients' mortality, with 5-year survival rates of only 22% (13% at 10 years) [2]. An increased risk of metastatic dissemination is exhibited by patients with Triple Negative BC (TNBC) [3]. It was also demonstrated that BC exhibits metastatic heterogeneity with distinct metastatic preferences to various organs, with the primary target organs being bone, lung, liver, and brain [4]. Revealing the underlying molecular mechanisms that dictate how TNBC cells escape the primary tumor, spread to distant organs, and initiate outgrowth at a distant site, is essential for developing therapies to prevent metastasis.

Research suggests that Protein kinase C eta (PKCη) promotes the invasive behavior of breast cancer and elevated levels of PKCη have been associated with increased tumor aggressiveness, and poor prognosis in some studies [5–8]. PKCη is a regulator of mammary gland development, and alterations in PKCη have been reported in BC tissues [9]. Several reports showed direct correlations between the expression of PKCη and invasive BC [10]. Moreover, it is reported that the increased expression of PKCη progressively enhances BC aggressiveness in the MCF10 breast cancer series [11, 12]. Studies suggest invasive breast tumors with high PKCη staining were associated with positive lymph node status [5]. However, more studies are needed to understand the better molecular mechanisms involved in PKCη to enhancing tumor metastasis. Here we demonstrate that inhibiting the expression or activity of PKCη may offer a strategy to impede TNBC growth and metastasis, suggesting that PKCη could present a potential therapeutic target in TNBC.

Recent studies from our laboratory have shown that the peptide encoded by a uORF in the 5' UTR of PKCη transcript has cytotoxic effects on specific cancers [13]. It has been observed that this uORF-encoded peptide (uPEP2) can inhibit cell proliferation, apoptosis induction, tumor growth suppression, and reduction of metastasis [13]. These effects of the peptide are mediated by the inhibition of PKCη kinase activity, resulting in reduced protein expression levels, thereby affecting various cellular processes, including cancer development and progression.

At the core of the intrinsic molecular machinery that enables tumor cells to disseminate from the primary tumor lesion is a reversible cellular state known as epithelial-to-mesenchymal transition (EMT) [14], through the loss of cell-cell adhesion molecules and the rearrangement of the cytoskeleton leading to the acquisition of highly invasive traits [15, 16]. Major features of the mesenchymal phenotype also include the loss of cell polarity and cell junctions [17]. As the expression of EMT-inducing genes increases, the cell surface changes dramatically; an important step be is the loss of E-cadherin, a transmembrane protein responsible for forming adherens junctions and a marker of the epithelial phenotype. Over the past decade, YAP and TAZ have emerged as important drivers of cancer development, tumor growth, and metastasis [18, 19], as increased YAP and TAZ expression or their nuclear localization in metastatic tumors (compared to primary tumors) in pancreatic cancer [20], breast cancer [21, 22], and prostate cancer was in strong correlation with poor prognosis [23].

In this study, we sought to investigate the potential role of PKCη in TNBC progression and metastasis. Through our experiments, we have demonstrated the crucial role of PKCη in the plasticity, invasiveness, and metastatic potential of TNBC cells. We have uncovered the mechanisms by which PKCη drives EMT and have shown that targeting PKCη is sufficient to reduce TNBC metastasis via the Hippo-YAP pathway. Here we also study the molecular mechanisms mediated by uPEP2 treatment of TNBC tumors. Our findings suggest that PKCη represents a promising therapeutic target for this highly lethal and metastatic disease.

PKCη depletion impairs TNBC cell migration and invasion

To elucidate the function of PKCη in metastatic TNBC, we used CRISPR/Cas9 genome editing to knockout PRKCH in 4T1 and MDA-MB-231 TNBC cell lines. The two clones obtained from each cell line demonstrated specific ablation of PKCη with no effect on other PKCs expressed in these cells (Fig. 1A; Supplementary Fig. S1A, S1B, S1C). Knockout of PKCη in 4T1 and MDA-MB-231 cells showed a minimal effect in inhibiting cell proliferation (reduction in ~ 15%) when compared to scrambled control cells (Fig. 1B; Supplementary Fig. S1D). In addition, a profound effect of PKCη was detected on cellular processes related to metastasis such as dissemination and intravasation (migration and invasion), survival in the circulation (anchorage-independent growth and anoikis prevention ability), and colonization (Fig. 1D; Supplementary Fig. S1E). The role of PKCη in promoting anchorage-independent growth was analyzed by soft agar colony formation assay to determine its role in promoting tumorigenesis (Fig. 1C). Our results demonstrated a significant reduction in the number and size of soft agar colonies in PKCη^KO 4T1 and MDA-MB-231 cells also showing less resistance to anoikis (Fig. 1C; Supplementary Fig. S1F).

We further show that PKCη is associated with the migration and invasion abilities of TNBC cell lines. Wound healing scratch assays were initially used to study the migration ability of 4T1 and MDA-MB-231 cells. Our results demonstrated lower wound closure rates for PKCη^KO cells compared to CRISPR control cells in these triple-negative cell lines (especially after 48h) (Fig. 1E, 1F). Using trans-well migration/invasion chamber assays, we also showed that ablation of PKCη significantly inhibits the migration of 4T1 and MDA-MB-231 cells, as the number of cells that migrated across the membrane was higher in the CRISPR control group compared with TNBC PKCη^KO cell lines (Fig. 1G, 1H). Collectively, our results demonstrate that PKCη could play a substantial role in the metastasis of TNBC.

Depletion of PKCη in TNBC cell lines inhibits malignancies progression and metastasis in mice.

To understand the role of tumor-derived PKCη on malignancy progression and metastasis we injected 4T1 PKCη^KO and CRISPR control cells orthotopically into the mammary fat pads of female immuno-deficient NSG mice. Mice were monitored for the development of primary xenograft tumors and sacrificed when tumors reached ~ 1000 mm³ in size. The tumors developed at the site of injection with 4T1 PKCη^KO cells were significantly smaller in size, and mass as compared to control tumors (Fig. 2A, 2B). At the end of the experiment, a comparison of relative organ weights between the two groups of mice injected with 4T1 PKCη^KO and CRISPR control cells depicted a significant reduction in the lungs weight of mice injected with 4T1-PKCη^KO cells (Fig. 2C). The presence of lung metastasis was seen as macro nodules in mice bearing 4T1-CRISPR-control tumors, while only a few metastases nodules were observed in lungs of mice xenografted with 4T1-PKCη^KO cells (Fig. 2D, 2E). Numerous micro and macro-nodules were frequently observed in the lungs and liver in all 4T1-CRISPR control-bearing mice compared to 4T1-PKCη^KO bearing mice as shown here by H&E staining (Fig. 2F, 2G).

To further strengthen the findings, MDA-MB-231 cells were genetically labeled with luciferase (Luc) to facilitate in vivo tracking. Both MDA-MB-231 PKCη^KO and CRISPR-control cells were orthotopically injected into the mammary fat pads of female NSG mice (n = 6). Mice were monitored for the development of primary xenograft tumors and the volume of primary tumors and body weight of mice were measured at two days intervals. A tumor growth delay of the primary tumor volumes was depicted in mice injected with PKCη^KO MDA-MB-231 cells compared to mice injected with CRISPR-control mice (Fig. 2H, 2I, 2J), although no significant difference was found in their relative organ weights (Supplementary Fig. S2A). The metastatic potential of MDA-MB-231 CRISPR-control and PKCη^KO was assessed by bioluminescent imaging in multiple mice organs. Higher bioluminescent imaging signals were observed in mice bearing CRISPR-control tumors compared to mice bearing-PKCη^KO cells (Fig. 2K). MDA-MB-231 CRISPR-control cells injected mice group exhibited multiple metastases in the liver, lungs, intestine, spleen, kidney, brain, and thoracic cavity, compared with significantly fewer metastases in MDA-MB-231 PKCη^KO cells injected mice group (Fig. 2L). Organs such as lungs, liver, and brain were collected, and the presence of micrometastasis was confirmed by H&E staining of tissue sections (Supplementary Fig. S2B, S2C, S2D).

PKCη contributes to epithelial-to-mesenchymal transition (EMT)

Our results demonstrated a role for PKCη in TNBC metastasis. Therefore, we examined whether it could promote EMT, one of the hallmarks of cancer metastasis associated with the acquisition of invasive and metastatic properties. Epithelial and mesenchymal cells can be distinguished by the expression of specific markers, such as E-cadherin for epithelial, whereas mesenchymal markers include the extracellular matrix component fibronectin and the intermediate filament protein vimentin [24].

We show that PKCη depletion in 4T1 led to the recovery of the epithelial marker E-cadherin (Fig. 3A), demonstrating that PKCη suppresses E-cadherin expression and suggesting that it plays a role in regulating cell-cell adhesion (Fig. 3B). Notably, the mesenchymal marker vimentin was markedly decreased in PKCη^KO cells, supporting a more epithelial-like phenotype for these cells. The epithelial marker EpCAM was also increased upon PKCη deletion. The increased expression of E-cadherin and the decreased expression of vimentin in 4T1 were further confirmed using confocal analysis (Fig. 3C, 3D). Furthermore, 4T1 PKCη^KO showed a decrease in the EMT marker ZEB1 compared to scrambled control cells (Fig. 3A, 3B). However, the EMT marker Slug was rather increased in 4T1 PKCη^KO compared to control cells, while Snail was unaffected by PKCη ablation. The absence of PKCη in MDA-MB-231 showed similar effects on the expression of the EMT markers ZEB1, Slug, and Snail, the epithelial marker EpCAM and the mesenchymal marker vimentin (Fig. 3B). However, it did not restore the expression of E-cadherin. The EMT characters of the 4T1 tumor samples were also decreased upon PKCη deletion (Supplementary Fig. S3D). From these results, it was evident that the presence of PKCη causes the induction of EMT behavior of tumor cells and promotes breast cancer metastasis.

PKCη promotes YAP stabilization and activation in TNBC

It was recently shown that YAP (Yes-associated transcriptional regulator) could be a key factor in determining the integrity of epithelial sheets, EMT, and also increase TNBC metastasis and poor prognosis, hence we hypothesized that PKCη might regulate YAP and thus affect TNBC plasticity [18, 25–28]. To explore that, we first analyzed the TNBC clinical samples from the TCGA and found that PKCη (PRKCH) and YAP1 mRNAs are positively correlated in breast cancer (ductal carcinoma) (Fig. 4A, 4B). High YAP1 or PRKCH levels correlate with poor prognosis of TNBC patients (Fig. 4C). Based on the association between YAP and PRKCH, we validated the protein expression in TNBC using western blotting and observed a decreased expression of YAP upon depletion of PKCη in both 4T1 and MDA-MB-231 (Fig. 4D, 4E). Since YAP and its ortholog transcriptional coactivator TAZ function as transcriptional coactivators are the effector proteins of the Hippo pathway, we quantified the levels of the key proteins in this pathway using western blotting. The results show that PKCη depletion specifically affected YAP/TAZ phosphorylation in both 4T1 PKCη^KO and MDA-MB-231 PKCη^KO; phosphorylation of YAP on S109 was decreased, while the phosphorylation on S127 and S397 residues was increased. The phosphorylation on the latter two residues determines the sequestration of YAP in the cytoplasm or its degradation, limiting its translocation to the nucleus to coactivate gene transcription [20, 21]. TAZ phosphorylation at S89 was also elevated upon PKCη depletion (Fig. 4D, 4E), implying cytoplasmic retention. Phosphorylation on S128, indicating YAP activation, was also significantly reduced upon PKCη depletion. Reduced YAP expression in PKCη^KO cells, most probably due to degradation, was further confirmed by immunofluorescence microscopy. The prominent YAP nuclear localization found in both CRISPR control cells was lost in PKCη^KO cells retaining only the cytoplasmic localization signal (Fig. 4F, 4G). Similar results were obtained for TAZ immunofluorescence in MDA-MB-231 (Supplimentary Fig. S3A). The absence of PKCη also resulted in diminished expression of YAP transcriptional targets such as AXL, pan-TEAD and CYR61 (Supplimentary Fig. S3B).

The immunohistochemistry analysis of PKCη, YAP, pYAP (S127), and Ki67 were performed in 4T1 and MDA-MB-231 xenografted tumor tissues (Fig. 4H, 4I). PKCη was primarily expressed in the cytoplasm. Its expression was significantly reduced in PKCη^KO xenografted tissue samples. Notably, a significant decrease in the expression of the cell proliferation marker Ki67 was depicted in PKCη^KO tumor samples, compared to CRISPR control (Fig. 4H, 4I). YAP expression was increased in both CRISPR control tumor tissues at the cytoplasm and nucleus but was significantly reduced in PKCη^KO cell xenografted tumor tissues. YAP phosphorylation on S127 in the cytoplasm was increased in both TNBC PKCη^KO xenografted tumors compared to CRISPR control tumors, in agreement with observed cytoplasmic retention.

PKCη is a novel regulator of YAP/TAZ activity.

We next sought to explore the molecular mechanisms by which PKCη regulates YAP. To examine the possibility of YAP binding to PKCη and to study their interaction, a protein-protein docking was performed. PKCη and YAP1 (TEAD binding domain) were docked (Fig. 5A), and the server predicted the docked pose at a confidence score of 0.86 (Range: 0–1) and docking score of -242.46. The confidence score is calculated using the formulae:

Confidence score = 1.0/[1.0 + e0.02*(Docking_Score + 150)]

When the confidence score is > 0.7 the two molecules are considered very likely to bind. Similarly, the more the negative score, the higher the possible binding model (Fig. 5A, 5B). The results from protein-protein docking predicted YAP1 to dominantly interact with the kinase domain of PKCη, which is mainly composed of α-helix and β-sheets. The interaction between YAP1 and PKCη is given in Supplimentary Fig. S4B. It was also observed that the S127 and S128 residues of YAP1 each formed four hydrogen bonds with amino acid residues Glu398, Arg405 and Glu 503 of PKCη. Both S127 and S128 are well reported to be crucial in cytoplasmic retention and activation of YAP1 (Fig. 5B). We anticipate that the interaction of YAP1 and PKCη may be responsible for the phosphorylation of S128 and the physical hindrance posed by the presence of PKCη prevent the phosphorylation of S127 by LATS and further interaction with any other proteins. The protection of S127 phosphorylation from LATS is further enhanced due to the presence of the α-helix structure of YAP (residue 61–73) and coiled structure (residue 116 − 123), which may act as a barrier to prevent any further access to the S127 site (Fig. 5B) thus, explaining and supporting the aforementioned hypothesis.

Considering the empirical nature of docking, next we used confocal microscopy to look for co-expression between PKCη and YAP in the TNBC cell lines, and found that both PKCη and YAP are co-localized in the cytoplasm and nucleus of the cells using immunofluorescence. In 4T1 cells, PKCη was present in the cytoplasm and nuclear envelope, while YAP was observed both in the cytoplasm and inside the nucleus. Their co-staining was depicted in the cytoplasm and nuclear envelope (Pearson coefficient 0.92 ± 0.2) (Fig. 5C). Staining of MDA-MB-231 cells also showed a co-localization of PKCη and YAP mainly in the cytoplasm (Pearson coefficient 0.94 ± 0.23) (Fig. 5C). We further verified complex formation between YAP and PKCη using pulldown experiments with YAP antibody immobilized beads. PKCη was associated with YAP-antibody conjugated beads in both 4T1 and MDA-MB-231 cell lysates, while immunoprecipitation by a control antibody did not show this association (Fig. 5D). We further confirmed this interaction by reverse pulldown of PKCη and showed the binding to YAP (Fig. 5E). Taken together, our data support direct interaction between PKCη and YAP1.

MST1 and LATS are the core components of the Hippo pathway kinase cascade, where MST1 phosphorylates LATS and LATS was shown to be phosphorylated by AKT [29]. YAP1 is phosphorylated on serine residues by LATS, especially on S127 and S397. Immunoblot analysis showed that the loss of PKCη resulted in increased AKT phosphorylation on S473 accompanied by increased pMST1 (Thr183) and pLATS1 (Thr1079) phosphorylation in both 4T1 and MDA-MB-231 cells compared to control cells (Fig. 5F). Moreover, YAP is phosphorylated at serine 127, creating a binding site for 14-3-3 proteins. The binding of 14-3-3 to phosphorylated YAP leads to the inactivation of YAP and its retention in the cytoplasm. The phosphorylation-dependent binding of YAP to 14-3-3 and its subsequent cytoplasmic retention regulate YAP activity and its downstream effects on cell behavior [12, 30]. This regulatory mechanism helps to reduce breast cancer cell migration and metastasis in TNBC. Using immunoprecipitation experiments, we show an association of pYAP S127 with 14-3-3ζ in 4T1 PKCη^KO (Fig. 5G, 5H), supporting this molecular mechanism.

A micro-peptide that degrades PKCη downregulates YAP activity in vitro and in mice.

We have recently reported that a peptide encoded by a short open reading frame upstream to the coding region of PKCη (uPEP2) inhibits the kinase activity of PKCη resulting also in reduced tumor growth and metastasis due to their degradation [13]. Therefore, we examined here if uPEP2 treatment, inhibiting the kinase activity of PKCη leading to its degradation, will also affect YAP. Treatment of 4T1 and MDA-MB-231 cells with uPEP2 significantly reduced the expression of PKCη and YAP (Fig. 6A). Moreover, the pattern of phosphorylation on YAP residues S127, S397, and S128 was very similar to that obtained upon PKCη knockout (Fig. 5A). uPEP2 significantly suppressed the levels of phosphorylated MST1 and LATS1, the upstream regulators of Hippo pathway (Fig. 6A, 6B) similar to PKCη^KO. An effect on the EMT markers ZEB1 and Snail was also evident in these cells upon uPEP2 treatment (Fig. 6A, 6B).

In an experiment conducted as previously described of xenografted MDA-MB-231 cells in NSG mice [13], IHC analysis of PKCη and YAP in MDA-MB-231 revealed that PKCη and YAP expression is significantly suppressed by uPEP2 treatment while YAP phosphorylation at S127 was increased (Fig. 6C). In these uPEP2 treated mice, micro-metastasis in liver and lungs were also reduced (Fig. 6D, 6E). Taken together, our results suggest that uPEP2 can be used in therapy to suppress TNBC growth and metastasis via the PKCη/YAP/Hippo pathway signaling axis.

TNBC is aggressive and highly metastatic, having fewer treatment options than other types of breast cancer [31, 32]. Thus, revealing the molecular mechanism mediating BC metastasis colud lead to new targets of intervention. In this study, we show the role of PKCη in promoting cell migration and invasion, and we demonstrate that its expression is required to maintain the mesenchymal state during EMT. Furthermore, we show that PKCη activates YAP/TAZ, tumor aggressiveness, and metastasis using in vitro and in vivo models. Our studies identify a novel role for PKCη in TNBC metastasis by regulating the Hippo pathway and demonstrating the function of PKCη in promoting EMT through activation of the YAP/TAZ in TNBC.

Our previous studies and others have demonstrated PKCη as an anti-apoptotic kinase that predicts poor prognosis in breast and lung cancer [8, 33, 34]. Its high expression in metastatic and aggressive tumors has been reported [8, 35]. However, the underlying molecular mechanism of PKCη in cancer metastasis is currently unknown [8]. Alterations in PKCη expression have been reported in human BC tissues [5]. PKCη expression was increased in locally invasive breast tumor tissues, and high levels of PKCη were detected in invasive tumors associated with significant lymph node metastases, suggesting a role for PKCη in cancer progression [5]. Here we show the function of PKCη in cell migration and promoting metastasis.

PKCη expression was ablated in 4T1 and MDA-MB-231 cells to study its function in malignancy progression and metastasis using orthotopic mouse models. 4T1 PKCη^KO cells and MDA-MB-231 PKCη^KO exhibited a minor reduction in cell proliferation, while the significant effect was on cell migration; PKCη^KO cells showed markedly reduced wound closure following wound formation. Using Boyden chambers, we also show that in the absence of PKCη expression, 4T1 and MDA-MB-231 cells exhibit decreased migration and invasion, suggesting a role for PKCη in cell migration. We further show that PKCη enhances cell survival and anoikis prevention ability, which have crucial roles in metastasis formation, allowing cancer cells to circulate and home to distant sites in the body. This is further substantiated by in vivo experiments with 4T1 xenografts in NSG mice, demonstrating that 4T1 PKCη^KO cells depicted reduced primary tumor size and a significant decrease in size and number of tumor metastases in the lungs, with fewer tumor nodules. MDA-MB-231 PKCη^KO xenografts depicted also reduced metastasis in distant organs of the brain, lung, and liver (Fig. 2).

EMT is one of the major reasons for the aggressive behavior of TNBC cells [36]. During EMT, epithelial cells lose their cell-to-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells-like populations leading to the initiation of BC metastasis [36, 37]. This is accompanied by altered gene expression profiles and the acquisition of specific markers characteristic of the epithelial and mesenchymal states. EMT transition leads to loss of E-cadherin and EpCAM [38] and prominent expression of vimentin, altering cell-cell adhesion and enhancing the migratory/invasion behavior [39]. Our study revealed a role for PKCη in the EMT program of TNBC; PKCη expression suppressed the epithelial markers E-cadherin and EpCAM and upregulated vimentin and ZEB1, acquiring a highly invasive phenotype (Fig. 3). PKCη promotes ZEB1 expression, does not affect Snail, but suppresses the expression of Slug suggesting partial EMT transition [25, 26, 40–42]. Recent studies indicate that EMT occurs in a gradual manner characterized by different levels of epithelial and mesenchymal markers, exhibiting intermediate characteristics referred to as partial EMT states. Recent reports suggest that partial EMT tumor cells contribute to metastasis formation. Cells undergoing partial EMT enhance collective cell invasion and are required for lung metastasis in breast cancer [40, 41].

Different signaling pathways were implicated in EMT, out of which the hippo pathway is the most studied in the context of EMT behavior and metastasis [43–45]. YAP/TAZ, two key downstream targets of the hippo pathway, frequently associated with human cancer metastasis, is negatively regulated by phosphorylation [19, 20]. As a transcriptional co-activator, YAP nuclear localization determines its activity [23]. Translocation of YAP/TAZ into the nucleus induces transcription of downstream genes through their binding with TEAD [46–48]. YAP/TAZ can also mediate the EMT process and contribute to the stemness maintenance of breast cancer cells. Moreover, YAP antagonizes E-cadherin to promote EMT in cancer cells [44, 49], thus enhancing metastasis.

Data analysis of BC patients with a high metastatic invasive propensity and low survival BC revealed a positive correlation between the elevated expression levels of PKCη and the expression of YAP/TAZ. Our experiments also show a direct correlation between the expression of PKCη and YAP; depletion of PKCη in both 4T1 and MDA-MB-231 resulted in decreased expression of YAP. Furthermore, YAP-regulated gene signature also correlated with PKCη expression. We demonstrate that PKCη regulates the phosphorylation on YAP/TAZ; phosphorylation of S127 and S397 of YAP, and S89 of TAZ are increased upon PKCη depletion culminating in reduced expression of YAP, and lower expression of YAP/TEAD-regulated proteins, including CTGF, CYR61, and AXL (Fig. 4D, 4E; Supplimentary Fig. S3B). MST and LATS phosphorylation cascade mediate S127 phosphorylation, where AKT serves as an upstream regulator of this signaling pathway. Depletion of PKCη activated the MST-LATS-YAP phosphorylation cascade through the hyper-activation/phosphorylation of AKT, leading to YAP retention in the cytoplasm and its degradation. Furthermore, phosphorylated YAP at S127 binds to 14-3-3 protein and remains in the cytoplasm for degradation, thus losing its function as a transcription cofactor [45]. Here we show that in the absence of PKCη, 14-3-3ζ was increased, accompanied by reduced YAP levels. Our results are consistent with reports showing that overexpression of YAP significantly reduced the expression of 14-3-3ζ [12, 49, 50]. Studies also show that patients with high YAP expression and low 14-3-3ζ expression have poor prognosis [12, 30]. We have previously reported that the PKCη is a negative regulator of AKT activity [51]. Here we also demonstrate that depletion of PKCη significantly enhances AKT activation as indicated by S473 phosphorylation leading to MST-LATS-YAP phosphorylation cascade and the phosphorylation on YAP S127 and S397. Hence, we show that PKCη is an upstream regulator of YAP promoting its activation and nuclear translocation.

Phosphorylation of YAP at S128, resulting in YAP activation, was implicated by nemo-like kinase (NLK) [52]. Dephosphorylated YAP, but with S128 phosphorylation, translocate into the nucleus and binds with TEAD to activate transcription of downstream genes that promote EMT and metastasis [47, 48]. However, in aggressive BC tissues, NLK expression was reported to be downregulated compared to corresponding normal tissues [53]. It was suggested that YAP phosphorylation at S128 inhibits S127 phosphorylation and 14-3-3 binding, thus overriding the inhibitory effects of the canonical Hippo pathway via S127 phosphorylation, leading to YAP activation and nuclear localization [45]. We show that in PKCη-depleted cells S128 is not phosphorylated. Moreover, we show the binding of PKCη and YAP (Fig. 5), supported by docking experiments indicating that PKCη can possibly bind to YAP (Fig. 5). Thus, our experiments imply a role for PKCη in YAP activation and its nuclear translocation; we propose that in the cytoplasm, PKCη mediates S128 phosphorylation of YAP, averts YAP phosphorylation at S127 and thereby binding of 14-3-3 protein, directing YAP to the nucleus to transcribe specific genes enhancing metastasis (Fig. 5) [52].

We have recently reported the identification of a kinase inhibitory peptide encoded by a short open reading frame (uORF) upstream to PKCη main coding sequence [13, 52]. We showed that this peptide (uPEP2) binds to PKCη and inhibits their kinase activity resulting in reduced cancer cell survival and migration [13]. Moreover, we demonstrated that uPEP2 effectively reduces MDA-MB-231-xenografted cancer tumor volumes and their metastasis to the lung and liver [13]. Here we show that treatment with uPEP2 of 4T1 and MDA-MB-231 cells also resulted in lower levels of YAP, accompanied by increased phosphorylation on S127 and S397, suggesting its cytoplasmic retention and degradation (Fig. 6E). Hence, the knockout of PKCη in breast cancer cells was phenocopied by the pharmacological treatment of these cells by uPEP2. Our findings provide further evidence that the uORF-encoded peptide could be utilized as a therapeutic agent for triple-negative breast tumors.

Collectively, our studies identify a novel role of PKCη as a negative regulator of the Hippo pathway and demonstrate its function in promoting EMT and metastasis through modulation of YAP/TAZ activity in TNBC. Our findings suggest that PKCη could represent a therapeutic target for this highly lethal and metastatic breast cancer.

AKT AKT Serine/Threonine Kinase

AXL AXL receptor tyrosine kinase

BC Breast Cancer

BRCA Breast Invasive Carcinoma

BSA Bovine Serum Albumin

CRISPR Clustered Regularly Interspaced Short Palindromic Repeats

CTGF Connective tissue growth factor

CYR61 Cysteine-rich angiogenic inducer 61

DAPI 4',6-diamidino-2-phenylindole

DH5α Douglas Hanahan 5 alpha

DMEM Dulbecco's Modified Eagle Medium

ECL Enhanced Chemiluminescence

EDTA Ethylenediamine Tetra Acetic acid

EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid)

EMT Epithelial-to-Mesenchymal Transitions

EpCAM Epithelial Cell Adhesion Molecule

FCS Fetal Calf Serum

gRNA guide Ribonucleic Acid

HR Hazard Ratio

HRP Horseradish Peroxidase

IB Immunoblot

IgG Immunoglobulin G

IgH Immunoglobulin Heavy Chain

IHC Immunohistochemistry

IP Immunoprecipitation

kDa kilodalton

Ki67 Kiel67

KMPLOT Kaplan-Meier plot

KO Knockout

LATS1 Large tumor suppressor kinase 1

mg milligram

mm millimeter

mRNAs Messenger Ribonucleic Acid

MST1 Macrophage Stimulating 1

NLK Nemo-Like Kinase

NSG NOD scid gamma mouse

OD Optical Density

OS Overall Survival

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

PKC Protein Kinase C

PKCα Protein kinase C alpha

PKCδ Protein kinase C delta

PKCε Protein kinase C epsilon

PKCζ Protein kinase C zeta

PKCη Protein kinase C eta

PRKCH Protein Kinase C eta

PVDF Polyvinylidene Difluoride

RIPA Radioimmunoprecipitation Assay

ROI Region of interest

SD Standard Deviation

SDS Sodium Dodecyl Sulphate

sgRNA Single guide RNA

TAZ Transcriptional coactivator with PDZ-binding motif

TBD Tead binding Domain

TCGA The Cancer Genome Atlas

TEAD TEA Domain Transcription Factor 1

TIMER Tumor Immune Estimation Resource.

TNBC Triple Negative Breast Cancer

TPM Transcript Per Million

uORF Upstream Open Reading Frames

uPEP2 encoded peptide from PKCeta uORF2

UTR Untranslated Regions

VSVG Vesicular stomatitis virus G protein

XTT (2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt

YAP1 Yes-Associated Protein 1

ZEB1 Zinc-finger E-box Binding Protein 1

β -Actin Beta-actin

Cell lines

4T1, PKCηKO 4T1, MDA-MB-231, PKCηKO MDA-MB-231, MDA-MB-231-Luc and PKCη^KO MDA-MB-231-Luc cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (no. 01–055-1A, Biological Industries). Media were supplemented with penicillin (100 units/mL), streptomycin (0.1 mg/mL), and L-glutamine (2 mM) plus 10% FBS for the cell lines. All cells were maintained in a 37°C, 5% CO₂ humidified incubator. No Mycoplasma contamination was detected with the Mycoplasma Detection Kit (PCR; HyLabs).

Peptides

uPEP2 (Myr-MASRGALRRCLSPGLPRLLHLSRGLA), was synthesized by GL-Biochem Ltd., Shanghai, China. The peptide contains a C-terminus amidation for stability and a myristoyl group, the N-terminus, to enable cell penetration.

PKCη knockout cell lines generation using CRISPR/Cas9 technology

In this study, we engineered the CRISPR-Cas9 Lentiviral system based on the protocol perfected by the Zhang lab [54, 55]. The PKCη gRNA sequence that targets exon 1 of the murine PRKCH gene (for 4T1) and non-specific scrambled sequences were designed using Benchling software and synthesized from Sigma, Israel, while the human PRKCH gene (for MDA-MB-231 and MDA-MB-231 Luc), and corresponding non-specific scrambled sequences were designed using Benchling software and synthesized from IDT-Syntezza, Israel (Table 1). The LentiCRISPRv2 vector was digested using BsmBI (NEB) and dephosphorylated using sheep Alkaline phosphatase for 30 mins at 37^oC. The digested plasmid was purified using the GeneJet Gel Extraction Kit (Thermo Scientific Inc., USA). The gRNA and scramble oligos were phosphorylated and annealed using T4 PNK (NEB) in T4 ligation buffer (Promega) in the following parameters: 37^oC - 30 min, 95^oC - 5 min, and ramp down to 25^oC at 5^oC/min. 50 ng BsmBI digested plasmid and 1:100 diluted oligos were ligated using T4 ligase (NEB) for 30 mins at RT. The ligation mix was transformed into DH5α, spread on Ampicillin agar plates, and incubated O/N at 37^oC. Positive clones were picked, and cultured and cloning was confirmed by colony PCR and sequencing. Plasmids were amplified using DH5α and purified by the MaxiPrep Plasmid DNA kit (Invitrogen, USA). LentiCRISPRv2Puro carrying PKCη gRNA or scrambled constructs were packaged into lentiviral particles using a mix of plasmids containing lentiviral packaging genes VSVG and psPAX2. The mixture and the specific plasmid were transfected into sub-confluent HEK293T by PEI reagent. The 293T cells were then maintained in DMEM medium supplemented with 10% FCS and antibiotics and incubated overnight at 37^oC and 5% CO₂. The medium was replaced 24 h after transfection, and 48 h later, the supernatants were collected, and cell debris was removed by centrifugation and passed through a 0.45 µm-filter. The viruses were concentrated using Amicon filter tubes (100kDA MWCO) (Merck Millipore, USA). A fresh culture of mouse 4T1 or human MDA-MB-231 and MDA-MB-231 Luc was then treated with the corresponding virus suspension mix containing 10 µg/ml protamine sulfate. Positive clones were isolated by Puromycin selection. The PRKCH deletion in the single-cell clones established were confirmed by sequencing and western blot.

Table 1

Mouse sgRNA for PRKCH	Fwd: 5’-CACCGCACAAAGTGGTCATAACCCAG-3’
Mouse scrambled sgRNA	5’-CACCGCACTACCAGAGCTAACTCA-3’
Human sgRNA for PRKCH	Fwd: 5’-CACCGCCACGCTCACCGTCAGATAG-3’
Human scrambled sgRNA	(5'-CACCGATCGTTTCCGCTTAACGGCG-3')
Sequencing primers
Mouse	Fwd: 5- ACCTGACGGTGAGCGTAGAC-3’ Rev: 5’- CTGTCGCGTCAACAGTGC-3’
Human	Fwd: 5’- ATGGCTATTTGAGGGTCCGC-3’ Rev: 5’- CGTTAGCGCAAAACTCCTCG-3’

Cell viability and proliferation assays.

4T1/MDA-MB-231 (CRISPR control and PKCη^KO) cells were seeded in 96-well plates (10,000 cells/well) and grown for 48 h. XTT (2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) assays were conducted according to the manufacturer's instructions (no. 20–300-1000, BI). The plates were read in a Multiskan Spectrum reader (Thermo Fisher Scientific). After 48 h to generate an optical density (OD) of cell number versus absorbance for all cell lines. Each experiment was repeated at least three times in triplicates.

For cell proliferation assays of PKCη knockout (PKCη^KO)/control (CRISPR/Cas9) 4T1 and MDA-MB-231 clones, cells were seeded (5000 cells/well) in 96-well plates. Cells were trypsinized, collected, and counted using an automated cell counter (Countess II FL, Thermo Scientific Inc.) at intervals of 24 h for five days. A minimum of four wells were prepared for each time point cell type. The data are representative of three independent experiments.

Colony formation assay

This assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. 4T1 and MDA-MB-231 (control and PKCη^KO) cells were detached and resuspended by trypsinization. Seed a total of 500 cells into each well of a 6-well plate. Incubate cells in a 5% CO₂environment at 37^oC. The control dishes for TNBC cells require 7 days to form sufficiently large clones. The experiment is repeated three times independently. At the end of the investigation, plates are washed in PBS, and cells are fixed (formaldehyde, 1%) and stained with a crystal violet solution (0.5 g crystal violet; 0.05% w/v). Wash dishes one at a time by dipping into the vessel of water in the sink. Air-dry dishes and digital images of the colonies are obtained using a camera. Colonies are counted using the imaging analysis software ImageJ.

Soft agar assay

4T1, MDA-MB-231 control, and PKCη^KO cells in complete growth medium with 0.6% agar were layered onto 1% agar beds in twelve-well plates; complete medium with 10% FBS were stained by added on top of cells and was replaced with fresh medium twice a week for 15 days. Cells by adding 200 μl of nitroblue tetrazolium chloride solution per well and incubating plates overnight at 37°C. Once colonies are stained, take photographs of wells using an imager and count colonies using image analysis software.

Anoikis Assay.

Anoikis resistance of tumor cell lines 4T1 and MDA-MB-231 (control and PKCη^KO) was determined by low adherence and re-attachment assay. Cells grown in flasks are detached using trypsin/EDTA and then seeded (5000 cells/well) in low attachment and normal attachment 96-well plates in media supplemented with 10% FBS. Cells are maintained in low adherence plates for 48 h and then transferred by pipetting the content of each well to normal adherence plates for 16 h. Cells that were seeded in a normal adherence plate are kept under these conditions for 48 hours plus an additional 16 hours. At the end of the assay, XTT (2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) assays were conducted according to the manufacturer’s instructions (no. 20–300-1000, BI). The plates were immediately read in a Multiskan Spectrum reader (Thermo Fisher Scientific). Cells plated in low attachment and in normal attachment 96 wells were assayed by XTT to generate an optical density (OD) of cell number versus absorbance for all cell lines. Each experiment was repeated at least three times in triplicates. The relative cell survival and re-attachment are calculated from the mean OD of normal attachment and the mean OD of low attachment plate.

Wound healing (Scratch) assays.

4T1, MDA-MB-231, and PKCη^KO cells were seeded in 24-well plates and grown to confluence. A gap in the confluent monolayer (middle of the well) was created using a sterile pipette tip (200 μL) followed by two PBS washes. Subsequently, medium containing low serum (0.1% FBS) was added, and the wound area were photographed (three images for each well at the indicated time points) at 4× magnification with an I X 70 Olympus Optical light microscope at different time point such as 0, 12, 24 and 48 h. The wound areas were measured and normalized to time 0 using ImageJ software (1.53i).

Invasion and migration assays.

The 4T1 and MDA-MB-231 cells (CRISPR control and PKCη^KO) cells (20,000 cells/well) were seeded into the upper chambers in the FBS-free DMEM medium. The lower chambers were loaded with 10% FBS DMEM medium. To assay invasive potential, 20,000 cells (4T1 / MDA-MB-231 cells; CRISPR control / PKCη^KO) were seeded into growth factor-reduced Matrigel invasion chambers (ThinCerts TM, 24 well, Greiner bio-one, 8 μm pore). After 24 h, cells were stained, with crystal violet solution. Cells on the lower chamber of the membrane were counted and quantified. Five fields of view were randomly chosen, and the average cell number was determined.

Co-expression, Correlation, and survival data Analysis

The co-expression data of PRKCH and YAP1 was generated from TCGA-BRCA dataset via the Morpheus platform at the Broad Institute (https://software.broadinstitute.org/morpheus/). The analysis was constrained to a cohort of 74 triple-negative cancer patients (invasive ductal carcinoma). The co-relation analysis of PRKCH and YAP1 was performed using TIMER 2.0 (http://timer.cistrome.org/) using Spearman's correlation [56]. The overall survival (OS) data of YAP1 and PRKCH were generated from KMPLOT webserver (https://kmplot.com/analysis/) using RNA-seq Geo Dataset GSE96058. The analysis was restricted to a patient cohort of 313 patients who had not received any endocrine or chemotherapy treatment.

Immunoblot analysis

Whole-cell extracts were prepared by lysing cells in a RIPA lysis buffer containing 10 mM Tris pH 8.0, 100 mM NaCl, 5 mM EGTA pH 8.0, 45 mM 2-mercaptoethanol, 1% NP-40, 10 mM EGTA pH 8.0, 50 mM NaF, and 0.1% SDS. Protease inhibitors (1 mM PMSF, 10 μg/ml aprotinine, and 10 μg/ml leupeptin) and phosphatase inhibitors (1 mM sodium orthovanadate, 50 mM β-glycerolphosphate, and 5 mM sodium pyrophosphate) were added just before cell lysis. The lysates were incubated on ice for 30 min and sheared several times through a 21-gauge needle. The cell lysates were centrifuged at 13,000 g for 25 min at 4^oC, and protein concentrations were determined by using the Bio-Rad (Hercules, CA) protein assay. Aliquots of 30–60 μg protein were prepared and resolved by electrophoresis on 10–15% polyacrylamide gels using Bio-Rad Mini-PROTEAN II cells. Proteins from the gel were electroblotted onto PVDF (Bio-Rad) in Bio-Rad Mini Trans-Blot transfer cells. After 1 h of blocking with 3% BSA in PBS at 37^oC, the PVDF membranes were incubated with the indicated primary antibodies overnight at 4^oC, followed by incubation with HRP-conjugated secondary antibodies. Immunoreactive protein bands were visualized using the ECL reagent (Biological Industries) and detected by the Gel Doc (Bio-Rad) system. Quantifications were accomplished by using ImageJ software. The antibodies used in this study were listed here: YAP (CST; #14074), MST1 (CST, #3682), pMST1 (CST, #49332), LATS1 (CST, #9153), 14-3-3 ζ/δ (CST, #7413), pLATS1 (CST, #9157). Phospho-YAP/TAZ antibody sampler kit, CST, #52420 [(pYAP (S127), pYAP (S109), pYAP (S397), pTAZ (S89), TAZ]. Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit, CST, #9782 [(Vimentin (D21H3), Snail (C15D3), Slug (C19G7), ZEB1 (D80D3), E-cadherin (24E10)]. pYAP1 (Ser128) (Thermo fisher scientific, Catalog # PA5-117264), PKCη (Santa Cruz, sc-101199); PKCη (Santa Cruz, sc-215). PKCε (610085, BD Biosciences), PKCδ (610398, BD Biosciences), PKCζ (ab108970, Abcam), PKCα (610108, BD Biosciences) EpCAM (CST, #93790), Ki67 (Abcam, Cat. No. ab32), and pAKT (Santa Cruz, sc-514032).

Protein – Protein Docking

To understand the interaction between the YAP1 and PKCη Protein-Protein Docking approach was used. Due to the lack of complete PKCη and YAP1-Tead binding Domain (TBD) crystal structure, a homology-based modeling approach was used [57]. The PKCη structure was generated using Swiss-modeler (https://swissmodel.expasy.org/). YAP1 -TEAD binding domain (TBD) was modelled using IntFOLD (https://www.reading.ac.uk/bioinf/IntFOLD/). The generated structures were validated using Swiss-model structure assessment (https://swissmodel.expasy.org/assess). Both the structures were prepared and optimized prior to docking. Any structural inconsistencies were also removed. The protein-protein docking was performed using online Hdock Server (http://hdock.phys.hust.edu.cn).

Co-immunoprecipitation

Co-IP of PKCη with mouse monoclonal antibody to YAP1

500µl of 3% BSA was used to precoat the Protein G beads (#17061801, Cytiva, Sweden). Protein G beads (200 μL) were pre-absorbed with mouse monoclonal anti-YAP1 (CST; #12395) (5µL/sample) for 90 mins in 4°C. Excess antibody was removed using wash buffer (25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100). PKCη CRISPR control cell lysates (2-2.5 mg/sample) of MDA-MB-231 or 4T1 were added to the bead-antibody complex, and incubated at 4°C overnight. Cells were lysed with RIPA buffer without SDS. Immune complexes were washed extensively with wash buffer, followed by the addition of SDS sample buffer and the corresponding samples were subsequently run on 10% Polyacrylamide gels and immunoblotted with anti-PKCη antibody (#ab179425, Abcam, UK). An anti-mouse IgG1 isotype control (#02-6100; Invitrogen, US) was also used simultaneously.

Reverse Co-IP of YAP1 with rabbit monoclonal antibody to PKCη

Following pre-coating with 3% BSA, Protein A beads (#sc-2001, Santa Cruz Biotechnology, USA) (200 μL) were pre-absorbed with anti-PKCη (#ab179425, Abcam, UK) (10µL/sample) for 90 mins in 4°C followed by a wash with wash buffer to remove unbound antibody. PKCη CRISPR control cell lysates (2-2.5 mg/sample) of MDA-MB-231 or 4T1 were added to the bead-antibody complex, and incubated at 4°C overnight. Immune complexes were processed (as mentioned above) and immunoblotted with an anti-YAP1 antibody. An anti-rabbit IgG isotype control (#ab37415; Abcam, UK) was also used simultaneously.

Co-IP of 14-33-3 with mouse monoclonal antibody to YAP1

Following pre-coating with 3% BSA, Protein G beads (200 μL) were pre-absorbed with mouse monoclonal anti-YAP1 (CST; #12395) (5µL/sample) for 90 mins in 4°C and excess antibody was removed using wash buffer. PKCη CRISPR control or knockout cell lysates (2-2.5 mg/sample) of 4T1 were added to the bead-antibody complex and incubated at 4°C overnight. Immune complexes were washed extensively with wash buffer, followed by the addition of SDS sample buffer, and the corresponding samples were subsequently run on 10% Polyacrylamide gels and immunoblotted with anti-14-3-3 antibody. An anti-mouse IgG1 isotype control was also used simultaneously.

Orthotopic Xenograft Breast Cancer Models in NSG Mice using 4T1 and MDA-MB-231 with and without PKCη.

Mice were housed in air-filtered laminar flow cabinets with a 12/12 h light-dark cycle and were fed food and water ad libitum. The mice were housed and handled according to the institutional guidelines of Ben-Gurion University of the Negev. The Institutional Animal Ethics Committee approved the animal experiments protocol. All animal experiments were conducted using 6- 8-wk-old female NSG mice (The Jackson Laboratory, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ). 4T1 (1×10⁶) / MDA-MB-231-Luc (4×10⁶) and PKCη^KO cells were suspended in PBS (100 μL) and injected subcutaneously in the mammary fat pad of NSG female mice [13]. Tumor volume was measured on alternate days using a digital Vernier caliper. The tumor volumes (mm3) were calculated using the following formula V = (W2 × L)/2 (V, tumor volume; W, tumor width; L, tumor length). Mice were sacrificed when control tumors (with PKCη) reached ∼10% of body weight (∼2,000 mm3). At the end of the experiment, all animals were weighed, euthanized, and liver, lung, spleen, kidney, brain, and tumors were harvested. The weights of tumors and relative organs weight were also recorded. Part of the tumors were preserved in 4% paraformaldehyde for histology and immunohistochemistry analysis. For immunoblot analysis, a small portion of the dissected tumors were snap-frozen in liquid nitrogen. Tumor tissue samples (25 mg) were homogenized in RIPA buffer (500 μL) containing protease inhibitors, followed by sonication for 5 minutes. Homogenized tissue samples were centrifuged at 13,000 × g for 25 min at 4°C, and supernatants were collected and subjected to immunoblot analysis.

In MDA-MB-231 xenografted model, the whole-body images of luciferase expression in NSG mice were monitored using the Xenogen In Vivo Imaging System (Xenogen). After anesthetizing the mice, 200 μL of D-luciferin (15 mg/mL) (no. LUCK-1G, Gold Biotechnology) was injected intraperitoneally, and in vivo, imaging analysis was performed. Luciferase expression data were quantified using Living Image software (4.7.3) in a fixed region of interest in Pixels.

Histological (H&E) staining

The paraffin-embedded tissues were then sectioned (5 μm) before being deparaffinized and rehydrated. Tissue sections were stained with hematoxylin for 3 min, washed with tap water for removing excess stain, counterstained with eosin for 30 s, dehydrated, and equilibrated with xylene. Tissues were then dehydrated and mounted (Surgipath, Sub-X, Mounting Medium).

Immunohistochemistry

Formalin-fixed, paraffin-embedded blocks with tumor samples were sectioned at a thickness of 5 μm using a fully automated rotary microtome (no. RM2255, Leica), dried for 1 h at 65°C, deparaffinized, and rehydrated. The slides were incubated in 10 mM citric acid buffer, pH 6 at 100°C for 20 min for antigen retrieval. The endogenous peroxidase activity was blocked with H₂O₂ (0.3%). Sections were then blocked for 1 h at room temperature with blocking solution (0.1% Tween, 5% bovine serum albumin) followed by incubation with primary antibody Ki67 (Abcam, Cat. No. ab32) (1:500), PKCη (1:50) (sc-215/sc-215-G), YAP (CST, #14074) (1:500) and pYAP S127 (CST, #4911) (1:500). The VECTASTAIN ABC Kits (no. PK-6200, Vector Laboratories, Inc.) was used for detection according to the manufacturer's protocol.

Images were captured by a PANNORAMIC MIDI scanner (3DHISTECH) and analyzed by QuPath software (0.2.1). The number of positive nuclei and the annotated area of each tissue of PKCη, YAP, pYAP S127, and Ki67 were calculated, and the results are denoted as the percentage of cells with positive nuclei using the software.

Immunofluorescence

Briefly, cells were fixed in 4% paraformaldehyde for 10 min, washed in PBS, permeabilized with Triton 0.1% for 10 min, and blocked in antibody diluent solution for 1 h. Antigen recognition was done by incubating with primary antibody PKCη (NBP2-38711, Novus Biologicals), YAP (CST, #14074), E-cadherin (CST, #14472), Vimentin (CST, #5741), TAZ (CST, #83669) overnight at 4^oC and with goat anti-mouse/rabbit Alexa Fluor 568/488/546/633 (Life Technologies) as secondary antibody for 1h at room temperature. Nuclei were counterstained with DAPI.

The coverslips were mounted on slides using DAKO mounting medium and imaged by Olympus FluoView FV1000 laser-scanning confocal microscope. Immunofluorescence images represent single 2-D confocal sections. The extent of colocalization of PKCη and YAP was quantified using the JACoP ImageJ plugin.

Statistical Analysis

The statistical significance of differences between experimental groups was determined using the unpaired two-tailed Student's t-test and the ANOVA test of variance in cases of multiple variables (using GraphPad Prism 5.2.1 software). P value < 0.05 was considered statistically significant.

Acknowledgments

This work was funded by the Israel Science Foundation (ISF, 2368/19 to Etta Livneh) and by (ISF, 302/21 to Moshe Eikabets) , The United States-Israel Binational Science Foundation (BSF, #2021055 to Moshe Eelkabets). Liju Vijayasteltar Belsamma is the recipient of a Kreitman post-doctoral fellowship from the Ben-Gurion University of the Negev, Beersheva, Israel.

Author contributions

L.V-B. Conceptualization, data curation, formal analysis, supervision, validation, investigation, visualization, writing, review and editing original draft. A.M. data curation, formal analysis, validation, investigation, editing original draft. D.R-J. data curation, formal analysis, validation, investigation, editing original draft. K.W. data curation, formal analysis, validation, editing original draft. S.J. validation, investigation, editing original draft. R.S. formal analysis, validation, investigation. E.E. validation, investigation. O.B. validation, investigation. M.E. Conceptualization, supervision, funding acquisition, writing, review and editing original draft, project administration. E.L. Conceptualization, supervision, funding acquisition, writing, review and editing original draft, project administration. All authors have read and approved the article.

Competing interests

The authors declare no competing interests.

Ethics declarations

The in vivo protocols and experiments were approved by the Institutional Animal Ethics Committee and by y the Israel Ministry of Health (Protocol # IL-17-03-2019(E)).

Availability of data

The dataset supporting the findings of this study is included in the article.

Smigiel JM, Taylor SE, Bryson BL, Tamagno I, Polak K, Jackson MW: Cellular plasticity and metastasis in breast cancer: a pre-and post-malignant problem. Journal of cancer metastasis and treatment 2019, 5.
Eng LG, Dawood S, Sopik V, Haaland B, Tan PS, Bhoo-Pathy N, Warner E, Iqbal J, Narod SA, Dent R: Ten-year survival in women with primary stage IV breast cancer. Breast cancer research and treatment 2016, 160:145-152.
Qiu J, Xue X, Hu C, Xu H, Kou D, Li R, Li M: Comparison of clinicopathological features and prognosis in triple-negative and non-triple negative breast cancer. Journal of cancer 2016, 7:167.
Liang Y, Zhang H, Song X, Yang Q: Metastatic heterogeneity of breast cancer: Molecular mechanism and potential therapeutic targets. In Seminars in cancer biology. Elsevier; 2020: 14-27.
Masso-Welch P, Winston J, Edge S, Darcy K, Asch H, Vaughan M, Ip M: Altered expression and localization of PKC eta in human breast tumors. Breast cancer research and treatment 2001, 68:211-223.
Raveh-Amit H, Hai N, Rotem-Dai N, Shahaf G, Gopas J, Livneh E: Protein kinase Cη activates NF-κB in response to camptothecin-induced DNA damage. Biochemical and biophysical research communications 2011, 412:313-317.
Karp G, Abu-Ghanem S, Novack V, Mermershtain W, Ariad S, Sion-Vardy N, Livneh E: Localization of PKCη in cell membranes as a predictor for breast cancer response to treatment. Oncology Research and Treatment 2012, 35:260-266.
Krasnitsky E, Baumfeld Y, Freedman J, Sion-Vardy N, Ariad S, Novack V, Livneh E: PKCη is a novel prognostic marker in non-small cell lung cancer. Anticancer research 2012, 32:1507-1513.
Caulfield J, Bolander F: Involvement of protein kinase C in mouse mammary gland development. Journal of endocrinology 1986, 109:29-34.
Basu A: The enigmatic protein kinase C-eta. Cancers 2019, 11:214.
Pal D, Basu A: The unique protein kinase Cη: Implications for breast cancer. International journal of oncology 2014, 45:493-498.
Zhang B, Gong A, Shi H, Bie Q, Liang Z, Wu P, Mao F, Qian H, Xu W: Identification of a novel YAP–14-3-3ζ negative feedback loop in gastric cancer. Oncotarget 2017, 8:71894.
Jayaram DR, Frost S, Argov C, Liju VB, Anto NP, Muraleedharan A, Ben-Ari A, Sinay R, Smoly I, Novoplansky O: Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs. Proceedings of the National Academy of Sciences 2021, 118:e2018899118.
Lu W, Kang Y: Epithelial-mesenchymal plasticity in cancer progression and metastasis. Developmental cell 2019, 49:361-374.
Martin TA, Ye L, Sanders AJ, Lane J, Jiang WG: Cancer invasion and metastasis: molecular and cellular perspective. In Madame Curie Bioscience Database [Internet]. Landes Bioscience; 2013
Desiniotis A, Kyprianou N: Significance of talin in cancer progression and metastasis. International review of cell and molecular biology 2011, 289:117-147.
Dongre A, Weinberg RA: New insights into the mechanisms of epithelial–mesenchymal transition and implications for cancer. Nature reviews Molecular cell biology 2019, 20:69-84.
Zanconato F, Cordenonsi M, Piccolo S: YAP/TAZ at the roots of cancer. Cancer cell 2016, 29:783-803.
Thompson BJ: YAP/TAZ: drivers of tumor growth, metastasis, and resistance to therapy. Bioessays 2020, 42:1900162.
Yang S, Zhang L, Purohit V, Shukla SK, Chen X, Yu F, Fu K, Chen Y, Solheim J, Singh PK: Active YAP promotes pancreatic cancer cell motility, invasion and tumorigenesis in a mitotic phosphorylation-dependent manner through LPAR3. Oncotarget 2015, 6:36019.
Bartucci M, Dattilo R, Moriconi C, Pagliuca A, Mottolese M, Federici G, Di Benedetto A, Todaro M, Stassi G, Sperati F: TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells. Oncogene 2015, 34:681-690.
Matteucci E, Maroni P, Luzzati A, Perrucchini G, Bendinelli P, Desiderio MA: Bone metastatic process of breast cancer involves methylation state affecting E-cadherin expression through TAZ and WWOX nuclear effectors. European Journal of Cancer 2013, 49:231-244.
Sun Z, Xu R, Li X, Ren W, Ou C, Wang Q, Zhang H, Zhang X, Ma J, Wang H: Prognostic value of yes-associated protein 1 (YAP1) in various cancers: a meta-analysis. PloS one 2015, 10:e0135119.
Heerboth S, Housman G, Leary M, Longacre M, Byler S, Lapinska K, Willbanks A, Sarkar S: EMT and tumor metastasis. Clinical and translational medicine 2015, 4:1-13.
Pastushenko I, Brisebarre A, Sifrim A, Fioramonti M, Revenco T, Boumahdi S, Van Keymeulen A, Brown D, Moers V, Lemaire S: Identification of the tumour transition states occurring during EMT. Nature 2018, 556:463-468.
Jolly MK, Boareto M, Huang B, Jia D, Lu M, Ben-Jacob E, Onuchic JN, Levine H: Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis. Front Oncol 2015, 5:155.
Park J, Kim D-H, Shah SR, Kim H-N, Kim P, Quiñones-Hinojosa A, Levchenko A: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases. Nature communications 2019, 10:2797.
Zanconato F, Battilana G, Cordenonsi M, Piccolo S: YAP/TAZ as therapeutic targets in cancer. Current opinion in pharmacology 2016, 29:26-33.
Oka T, Mazack V, Sudol M: Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP). Journal of Biological Chemistry 2008, 283:27534-27546.
Moon S, Kim W, Kim S, Kim Y, Song Y, Bilousov O, Kim J, Lee T, Cha B, Kim M: Phosphorylation by NLK inhibits YAP‐14‐3‐3‐interactions and induces its nuclear localization. EMBO reports 2017, 18:61-71.
Cleator S, Heller W, Coombes RC: Triple-negative breast cancer: therapeutic options. The lancet oncology 2007, 8:235-244.
Roshanzamir F, Robinson JL, Cook D, Karimi-Jafari MH, Nielsen J: Metastatic triple negative breast cancer adapts its metabolism to destination tissues while retaining key metabolic signatures. Proceedings of the National Academy of Sciences 2022, 119:e2205456119.
Zurgil U, Ben-Ari A, Atias K, Isakov N, Apte R, Livneh E: PKCη promotes senescence induced by oxidative stress and chemotherapy. Cell death & disease 2014, 5:e1531-e1531.
Tamarkin A, Zurgil U, Braiman A, Hai N, Krasnitsky E, Maissel A, Ben-Ari A, Yankelovich L, Livneh E: DNA damage targets PKCη to the nuclear membrane via its C1B domain. Experimental cell research 2011, 317:1465-1475.
Karp G, Maissel A, Livneh E: Hormonal regulation of PKC: estrogen up-regulates PKCη expression in estrogen-responsive breast cancer cells. Cancer letters 2007, 246:173-181.
Sun X, Wang M, Wang M, Yao L, Li X, Dong H, Li M, Li X, Liu X, Xu Y: Exploring the Metabolic Vulnerabilities of Epithelial–Mesenchymal Transition in Breast Cancer. Frontiers in Cell and Developmental Biology 2020, 8:655.
Liu X, Li J, Cadilha BL, Markota A, Voigt C, Huang Z, Lin PP, Wang DD, Dai J, Kranz G: Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis. Science advances 2019, 5:eaav4275.
Hyun K-A, Koo G-B, Han H, Sohn J, Choi W, Kim S-I, Jung H-I, Kim Y-S: Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer. Oncotarget 2016, 7:24677.
Usman S, Waseem NH, Nguyen TKN, Mohsin S, Jamal A, Teh M-T, Waseem A: Vimentin is at the heart of epithelial mesenchymal transition (EMT) mediated metastasis. Cancers 2021, 13:4985.
Pastushenko I, Blanpain C: EMT transition states during tumor progression and metastasis. Trends in cell biology 2019, 29:212-226.
Campbell K, Rossi F, Adams J, Pitsidianaki I, Barriga FM, Garcia-Gerique L, Batlle E, Casanova J, Casali A: Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors. Nature communications 2019, 10:2311.
Lüönd F, Sugiyama N, Bill R, Bornes L, Hager C, Tang F, Santacroce N, Beisel C, Ivanek R, Bürglin T: Distinct contributions of partial and full EMT to breast cancer malignancy. Developmental cell 2021, 56:3203-3221. e3211.
Feldker N, Ferrazzi F, Schuhwerk H, Widholz SA, Guenther K, Frisch I, Jakob K, Kleemann J, Riegel D, Bönisch U: Genome‐wide cooperation of EMT transcription factor ZEB 1 with YAP and AP‐1 in breast cancer. The EMBO journal 2020, 39:e103209.
Beavon I: The E-cadherin–catenin complex in tumour metastasis: structure, function and regulation. European journal of cancer 2000, 36:1607-1620.
Hong W, Guan K-L: The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway. In Seminars in cell & developmental biology. Elsevier; 2012: 785-793.
Yuan Y, Li D, Li H, Wang L, Tian G, Dong Y: YAP overexpression promotes the epithelial-mesenchymal transition and chemoresistance in pancreatic cancer cells. Molecular medicine reports 2016, 13:237-242.
Zhao W, Wang M, Cai M, Zhang C, Qiu Y, Wang X, Zhang T, Zhou H, Wang J, Zhao W: Transcriptional co-activators YAP/TAZ: Potential therapeutic targets for metastatic breast cancer. Biomedicine & Pharmacotherapy 2021, 133:110956.
Lamar JM, Stern P, Liu H, Schindler JW, Jiang Z-G, Hynes RO: The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proceedings of the National Academy of Sciences 2012, 109:E2441-E2450.
Lu J, Guo H, Treekitkarnmongkol W, Li P, Zhang J, Shi B, Ling C, Zhou X, Chen T, Chiao PJ: 14-3-3ζ cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Cancer cell 2009, 16:195-207.
Lu H-C, Chou F-P, Yeh K-T, Chang Y-S, Hsu NC, Chang J-G: Analysing the expression of protein kinase C eta in human hepatocellular carcinoma. Pathology 2009, 41:626-629.
Shahaf G, Rotem-Dai N, Koifman G, Raveh-Amit H, Frost SA, Livneh E: PKCη is a negative regulator of AKT inhibiting the IGF-I induced proliferation. Experimental cell research 2012, 318:789-799.
Hong AW, Meng Z, Yuan HX, Plouffe SW, Moon S, Kim W, Jho Eh, Guan KL: Osmotic stress‐induced phosphorylation by NLK at Ser128 activates YAP. EMBO reports 2017, 18:72-86.
Huang Y, Jiang Y, Lu W, Zhang Y: Nemo-like kinase associated with proliferation and apoptosis by c-Myb degradation in breast cancer. PloS one 2013, 8:e69148.
Sanjana NE, Shalem O, Zhang F: Improved vectors and genome-wide libraries for CRISPR screening. Nature methods 2014, 11:783-784.
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG: Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014, 343:84-87.
Li T, Fu J, Zeng Z, Cohen D, Li J, Chen Q, Li B, Liu XS: TIMER2. 0 for analysis of tumor-infiltrating immune cells. Nucleic acids research 2020, 48:W509-W514.
Yan Y, Tao H, He J, Huang S-Y: The HDOCK server for integrated protein–protein docking. Nature protocols 2020, 15:1829-1852.

No competing interests reported.

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YAP activation and Hippo signaling suppression by PKC eta promote triple-negative breast cancer metastasis

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Abstract

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Introduction

Results

PKCη depletion impairs TNBC cell migration and invasion

PKCη promotes YAP stabilization and activation in TNBC

Discussion

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