Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21Cip1 expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed–Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21Cip1 and p16INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated β-galactosidase (SA-β-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21Cip1, p16INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed ‘senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

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The successful treatment of cancer in a disseminated stage using chemotherapy is limited by the occurrence of drug resistance, often mediated by anti-apoptotic mechanisms. Thus the challenge is to pinpoint the underlying key factors and to develop therapies for their direct targeting. Protein kinase C (PKC) enzymes are promising candidates, as some PKCs were shown to be involved in regulation of apoptosis. Our studies and others have shown that PKCη is an anti-apoptotic kinase, able to confer protection on tumour cells against stress and chemotherapy. We have demonstrated that PKCη shuttles between the cytoplasm and the nucleus and that upon DNA damage is tethered at the nuclear membrane. The C1b domain mediates translocation of PKCη to the nuclear envelope and, similar to the full-length protein, could also confer protection against cell death. Furthermore, its localization in cell and nuclear membranes in breast cancer biopsies of neoadjuvant-treated breast cancer patients was an indicator for poor survival and a predictor for the effectiveness of treatment. PKCη is also a novel biomarker for poor prognosis in non-small-cell lung cancer (NSCLC). Thus PKCη presents a potential target for therapy where inhibition of its activity and/or translocation to membranes could interfere with the resistance to chemotherapy.