The PKC pathway participates in the aberrant accumulation of Fra-1 protein in invasive ER-negative breast cancer cells

Karine BELGUISE¹, Sandrine MILORD¹, Florence Galtier¹, Gabriel MOQUET- TORCY², Marc Piechaczyk² and Dany Chalbos¹.

 

Activating protein 1 (AP-1) activity, which is induced by a vast number of extracellular stimuli, such as growth factors, cytokines, tumour promoters and environmental stresses, has diverse biological functions and plays critical roles in regulating cell growth, differentiation, apoptosis, development and tumourigenesis¹. The AP-1 transcription factor is a dimeric protein complex comprising primarily Jun and Fos family members. While Jun proteins (c-Jun, JunB, and JunD) form homodimers or heterodimers with Fos proteins (c-Fos, Fra-1, Fra-2 and FosB), Fos proteins cannot associate with each other. Jun-Fos heterodimers interact more stably than Jun-Jun homodimers and therefore control transcription more efficiently. The combinatorial diversity of the dimers varies with the expression and activation levels of the individual components according to the cell type, the environmental situation and the phase of the cell cycle, suggesting that the various dimers display different properties and functions².

Emerging evidence suggests an important role for Fra-1 in oncogenesis and the progression or maintenance of many tumour types^3,4. Fra-1 has been shown to be a mediator of the Ras-induced transformation of NIH3T3 cells and thyroid cells^5,6. Whereas Fra-1 is constitutively expressed in a limited number of tissues, a high Fra-1 concentration is found in numerous cancer cell lines and tissues, including thyroid, breast, lung, brain, endometrial, prostate, bladder and colon carcinomas. Furthermore, in these different models, the manipulation of the Fra-1 concentration has indicated the active role of Fra-1 in the maintenance or acquisition of a more aggressive phenotype ^7-12.

In breast cancer, the most invasive cell lines have high AP-1 DNA-binding activity that is mostly due to Fra-1-containing heterodimers¹³. Aberrant Fra-1 protein levels have been detected in cells expressing neither oestrogen a (ER) nor progesterone receptors (PR) and expressing a number of mesenchymal markers. Moreover, Fra-1 plays an active role in breast cancer cell growth, invasion, motility and the control of cell morphology^7,10. Altogether, these data suggest that Fra-1 could be involved in breast cancer progression. In agreement with these studies, Fra-1 expression has been associated with hyperplastic and neoplastic proliferative breast disorders^14-17. Moreover, the tumour cell-induced de novo overexpression of Fra-1 in macrophages has recently been suggested to play a role in the immunosuppressive mechanisms correlated with mammary tumour progression¹⁸.

Fra-1 is regulated at the transcriptional level through numerous extracellular stimuli. However, Fra-1 is an intrinsically unstable protein and the regulation of its stability may be fundamental for its accumulation⁴. Fra-1 is among the most upregulated targets under Ras transformation conditions and its accumulation depends on both transcriptional auto-regulation and ERK‑dependent post-translational stabilisation. Indeed, the Fra-1 half-life is increased upon ERK1/2 pathway activation in thyroid¹⁹ and colon tumours^20,21. The ERK1/2 pathway has been shown to lead to the phosphorylation of serines S252 and S265, thereby inhibiting Fra-1 degradation during both normal physiological induction and the constitutive activation of this cascade in human colon cancer cells expressing oncogenic forms of KRAS and BRAF, which both activate ERK. However, because Ras mutations are not frequent in breast cancer cells²², we hypothesised that other kinases might play a role in the aberrant accumulation of hyperphosphorylated Fra-1 in these cells. Here, we tested the role of the PKCq pathway.

PKCq is a novel PKC that is activated by diacylglycerol but not by calcium²³. This serine/threonine kinase is a critical component of the immune system, in which it controls T lymphocyte fate and function²⁴. PKCq is overexpressed in gastrointestinal stromal tumours²⁵ and has only recently been implicated in breast cancers. PKCq has been reported to promote c-Rel-driven mammary tumourigenesis in mice by repressing ERα synthesis²⁶. In addition, the PKCq protein stimulates the proliferation and motility of breast cancer cells and is detectable and present in an active form only in ER-negative (ER-) breast cancer cells. Along the same line, ER- tumours in patients express an elevated level of PKCq mRNA compared to ER+ tumours²⁷.

We report here that high PKCq activity leads to a strong expression of Fra-1 in ER- invasive breast cancer cell lines. PKCq acts through the activation of ERK1/2 and Ste20-related proline-alanine-rich kinase (SPAK) pathways and stabilises Fra-1 protein by inducing its phosphorylation on S265, T223 and T230. Moreover, the high accumulation of Fra-1 induced by the PKCq pathway is critical to mediate the effect of this kinase on cell migration.

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Here, we report that a high level of hyperphosphorylated Fra-1 can be found in invasive breast cancer cell lines independently of activated ERK1/2 level, indicating that other pathways are likely to be involved in Fra-1 phosphorylation and stabilisation. We addressed the role of PKCq in Fra-1 accumulation, as it has recently been implicated in breast cancer²⁶ and is activated in invasive ER- breast cancer. We show that the PKCq pathway participates in Fra-1 stabilisation through the activation of both ERK1/2 and SPAK pathways.

Although both pathways are activated by PKCq in all of the breast cancer cells we tested, their relative contributions to the accumulation of Fra-1 appeared to be different depending on the cellular context (activation levels of PKCq and ERK1/2). Fra-1 stabilisation by PKCq in ER+ MCF7 cells requires equally the stimulation of both ERK1/2 and SPAK activities. However, in ER- MDA-MB231 cells, which display high ERK1/2 activity, the action of PKCθ mainly goes through this MAPK, while in ER- BT549 cells which show moderate ERK1/2 activity, its effect tends to be the result of SPAK activation. Interestingly, MDA-MB231 cells carry oncogenic constitutively active forms of K-Ras and B-Raf whereas BT549 cells express wild-type form of Ras and Raf^29,30. Morevover, high Fra-1 levels were detected in ER- breast cancer cells harbouring (Hs578T and MDA-MB231) or not (MDA-MB436, BT549, HCC38 and MDA-MB157) Ras pathway activating mutations. Our data suggest that the strong Fra-1 expression results not only from the stimulation of Ras-ERK1/2 pathway, but also from the activation of PKCθ-ERK1/2 and PKCθ-SPAK pathways in a cell-dependent manner.

We show that the phosphorylation of S265, T223 and T230 is crucial for Fra-1 stabilisation by the PKCq pathway. S265 is the main residue responsible for ERK1/2-driven Fra-1 stabilisation and, by analogy with c-Fos, is likely a target for ERK1 and/or ERK2 (ref. 21). Interestingly, SPAK has been reported to be crucial for the induction of PKCθ-mediated AP-1 activity in T lymphocytes²⁸, but the effector(s) downstream of SPAK is unknown. It is therefore tempting to speculate that this factor is a Fra-1-containing AP-1 dimer. We do not know yet whether Fra-1 is a direct substrate of SPAK, and other kinases may act downstream of SPAK. Fra-1 does not possess the consensus motif [S/G/V]RFx[V/I]xx[V/I/T/S]xx³¹ shown to mediate the binding of SPAK to its known substrates, such as members of the cation chloride co-transporters superfamily³² and the TNF receptor RELT³³. In addition, we cannot exclude a direct effect of PKCq on Fra-1, and other kinases could act downstream of PKCθ. Only a few PKCq substrates or potential candidate substrates, such as moesin, SPAK and CARMA1 (ref. 23), are known, and there is no described consensus sequence of phosphorylation by PKCq.

While both Fra-1 and PKCθ expressions are inversely correlated with the ERa status of breast cancer cells, activated PKCθ level does not systematically follow Fra-1 level among the different cell lines (Figure 1A). PKCθ is a strong inducer of Fra-1 expression; however according to the literature, it is not the only one³ and PKCθ-independent regulators of FOSL1 mRNA synthesis and Fra-1 protein stability may be differently expressed depending on the ER- cell lines. Moreover, PKCθ activation in T lymphocytes requires diacylglycerol and at least phosphorylation of T538 and Y9034 . In our study, PKCθ activation was measured by T538 phosphorylation and it would be interesting to evaluate the phosphorylation level of Y90 to determine whether it could correlate better with Fra-1 expression. Currently, PKCθ activation is not well understood and other activating phosphorylation sites not yet discovered could be critical in breast cancer. The 3-phosphoinositide-dependent kinase 1 (PDK1) and the src family protein tyrosine kinase Lck have been proposed to phosphorylate PKCθ on residues 538 and 90, respectively³⁴. Recently, the GCK-like kinase (MAP4K3) has been shown to directly phosphorylate PKCθ on T538 (ref. 035). While expression of GLK has not been studied in breast cancer, both LCK³⁶ and PDK1 (ref. 37) have been reported to be overexpressed, especially in ER- compared to ER+ tumours for the tyrosine kinase. Therefore, further studies in breast cancer field are required to discover the factors regulating PKCθ activation, including the enzymes responsible for the accumulation of diacylglycerol and/or the kinase(s) phosphorylating PKCθ.

Fra-1 is an important target of the PKCq pathway in breast cancer cells because PKCq-driven Fra-1 expression mediates PKCq effects on cell migration and invasion. Interestingly, we found that Fra-2, which also enhances cell invasion, was also regulated by the PKCq pathway (not shown). However, Fra-2-induced invasion occurs through a different mechanism involving de novo RelB synthesis³⁸. Fra-2 could therefore also participate in the enhancement of cell invasion induced by the kinase.

It is noteworthy that Fra-1 has recently been implicated in the epithelial-mesenchymal transition (EMT) of mammary cells by regulating slug expression³⁹, ZEB1/2 expression⁴⁰ or the neo-synthesis of microRNAs miR-221 and miR-222, which are associated with the basal-like subtype of breast cancer⁴¹. Fra-1 is a critical and necessary downstream target of ERK for the induction of EMT and the acquisition of a motile and invasive profile in non-tumourigenic epithelial cells^40,42. Conversely, Fra-1 extinction increases the expression of epithelial genes and decreases the levels of mesenchymal markers in the Ras-mutated MDA-MB231 cell line³⁹. We could therefore speculate that activated PKCq may also induce EMT in cells in which it is aberrantly expressed.

In summary, our findings identify PKCθ as an important regulator of Fra-1 accumulation in ER- basal-like breast cancer cells and suggest that PKCq may participate in progression of some breast cancers. As a consequence, it may be important to assess the potential effect of PKCq in the progression of other cancers in which Fra-1 has been associated with a more aggressive phenotype.