CDK4

MAP3K5 phosphorylates and activates MAP2K3 (MKK3) and MAP2K6 (MKK6) (Ichijo et al. 1997, Takekawa et al. 2005), which act as p38 MAPK kinases, as well as MAP2K4 (SEK1) (Ichijo et al. 1997, Matsuura et al. 2002), which, together with MAP2K7 (MKK7), acts as a JNK kinase.

MKK3 and MKK6 phosphorylate and activate p38 MAPK alpha (MAPK14) and beta (MAPK11) (Raingeaud et al. 1996), enabling p38 MAPKs to phosphorylate and activate MAPKAPK2 (MK2) and MAPKAPK3 (MK3) (Ben-Levy et al. 1995, Clifton et al. 1996, McLaughlin et al. 1996, Sithanandam et al. 1996, Meng et al. 2002, Lukas et al. 2004, White et al. 2007), as well as MAPKAPK5 (PRAK) (New et al. 1998 and 2003, Sun et al. 2007).

Phosphorylation of JNKs (MAPK8, MAPK9 and MAPK10) by MAP3K5-activated MAP2K4 (Deacon and Blank 1997, Fleming et al. 2000) allows JNKs to migrate to the nucleus (Mizukami et al. 1997) where they phosphorylate JUN. Phosphorylated JUN binds FOS phosphorylated by ERK1 or ERK2, downstream of activated RAS (Okazaki and Sagata 1995, Murphy et al. 2002), forming the activated protein 1 (AP-1) complex (FOS:JUN heterodimer) (Glover and Harrison 1995, Ainbinder et al. 1997).

Activation of p38 MAPKs and JNKs downstream of MAP3K5 (ASK1) ultimately converges on transcriptional regulation of CDKN2A locus. In dividing cells, nucleosomes bound to the CDKN2A locus are trimethylated on lysine residue 28 of histone H3 (HIST1H3A) by the Polycomb repressor complex 2 (PRC2), creating the H3K27Me3 (Me3K-28-HIST1H3A) mark (Bracken et al. 2007, Kotake et al. 2007). The expression of Polycomb constituents of PRC2 (Kuzmichev et al. 2002) - EZH2, EED and SUZ12 - and thereby formation of the PRC2, is positively regulated in growing cells by E2F1, E2F2 and E2F3 (Weinmann et al. 2001, Bracken et al. 2003). H3K27Me3 mark serves as a docking site for the Polycomb repressor complex 1 (PRC1) that contains BMI1 (PCGF4) and is therefore named PRC1.4, leading to the repression of transcription of p16-INK4A and p14-ARF from the CDKN2A locus, where PCR1.4 mediated repression of p14-ARF transcription in humans may be context dependent (Voncken et al. 2005, Dietrich et al. 2007, Agherbi et al. 2009, Gao et al. 2012). MAPKAPK2 and MAPKAPK3, activated downstream of the MAP3K5-p38 MAPK cascade, phosphorylate BMI1 of the PRC1.4 complex, leading to dissociation of PRC1.4 complex from the CDKN2A locus and upregulation of p14-ARF transcription (Voncken et al. 2005). AP-1 transcription factor, formed as a result of MAP3K5-JNK signaling, as well as RAS signaling, binds the promoter of KDM6B (JMJD3) gene and stimulates KDM6B expression. KDM6B is a histone demethylase that removes H3K27Me3 mark i.e. demethylates lysine K28 of HIST1H3A, thereby preventing PRC1.4 binding to the CDKN2A locus and allowing transcription of p16-INK4A (Agger et al. 2009, Barradas et al. 2009, Lin et al. 2012).

p16-INK4A inhibits phosphorylation-mediated inactivation of RB family members by CDK4 and CDK6, leading to cell cycle arrest (Serrano et al. 1993). p14-ARF inhibits MDM2-mediated degradation of TP53 (p53) (Zhang et al. 1998), which also contributes to cell cycle arrest in cells undergoing oxidative stress. In addition, phosphorylation of TP53 by MAPKAPK5 (PRAK) activated downstream of MAP3K5-p38 MAPK signaling, activates TP53 and contributes to cellular senescence (Sun et al. 2007)

Here, transcriptional regulatory processes that mediate the SASP are annotated. DNA damage triggers ATM-mediated activation of TP53, resulting in the increased level of CDKN1A (p21). CDKN1A-mediated inhibition of CDK2 prevents phosphorylation and inactivation of the Cdh1:APC/C complex, allowing it to ubiquitinate and target for degradation EHMT1 and EHMT2 histone methyltransferases. As EHMT1 and EHMT2 methylate and silence the promoters of IL6 and IL8 genes, degradation of these methyltransferases relieves the inhibition of IL6 and IL8 transcription (Takahashi et al. 2012). In addition, oncogenic RAS signaling activates the CEBPB (C/EBP-beta) transcription factor (Nakajima et al. 1993, Lee et al. 2010), which binds promoters of IL6 and IL8 genes and stimulates their transcription (Kuilman et al. 2008, Lee et al. 2010). CEBPB also stimulates the transcription of CDKN2B (p15-INK4B), reinforcing the cell cycle arrest (Kuilman et al. 2008). CEBPB transcription factor has three isoforms, due to three alternative translation start sites. The CEBPB-1 isoform (C/EBP-beta-1) seems to be exclusively involved in growth arrest and senescence, while the CEBPB-2 (C/EBP-beta-2) isoform may promote cellular proliferation (Atwood and Sealy 2010 and 2011). IL6 signaling stimulates the transcription of CEBPB (Niehof et al. 2001), creating a positive feedback loop (Kuilman et al. 2009, Lee et al. 2010). NF-kappa-B transcription factor is also activated in senescence (Chien et al. 2011) through IL1 signaling (Jimi et al. 1996, Hartupee et al. 2008, Orjalo et al. 2009). NF-kappa-B binds IL6 and IL8 promoters and cooperates with CEBPB transcription factor in the induction of IL6 and IL8 transcription (Matsusaka et al. 1993, Acosta et al. 2008). Besides IL6 and IL8, their receptors are also upregulated in senescence (Kuilman et al. 2008, Acosta et al. 2008) and IL6 and IL8 may be master regulators of the SASP.

IGFBP7 is also an SASP component that is upregulated in response to oncogenic RAS-RAF-MAPK signaling and oxidative stress, as its transcription is directly stimulated by the AP-1 (JUN:FOS) transcription factor. IGFBP7 negatively regulates RAS-RAF (BRAF)-MAPK signaling and is important for the establishment of senescence in melanocytes (Wajapeyee et al. 2008).

Please refer to Young and Narita 2009 for a recent review

MAP kinases MAPK1 (ERK2) and MAPK3 (ERK1), which are activated by RAS signaling, phosphorylate ETS1 and ETS2 transcription factors in the nucleus (Yang et al. 1996, Seidel et al. 2002, Foulds et al. 2004, Nelson et al. 2010). Phosphorylated ETS1 and ETS2 are able to bind RAS response elements (RREs) in the CDKN2A locus and stimulate p16-INK4A transcription (Ohtani et al. 2004). At the same time, activated ERKs (MAPK1 i.e. ERK2 and MAPK3 i.e. ERK1) phosphorylate ERF, the repressor of ETS2 transcription, which leads to translocation of ERF to the cytosol and increased transcription of ETS2 (Sgouras et al. 1995, Le Gallic et al. 2004). ETS2 can be sequestered and inhibited by binding to ID1, resulting in inhibition of p16-INK4A transcription (Ohtani et al. 2004).Transcription of p14-ARF is stimulated by binding of E2F transcription factors (E2F1, E2F2 or E2F3) in complex with SP1 to p14-ARF promoter (Parisi et al. 2002).Oncogenic RAS signaling affects mitochondrial metabolism through an unknown mechanism, leading to increased generation of reactive oxygen species (ROS), which triggers oxidative stress induced senescence pathway. In addition, increased rate of cell division that is one of the consequences of oncogenic signaling, leads to telomere shortening which acts as another senescence trigger

Destruction of the D type cyclins accompanies the end of the G1 phase, and the E type cyclins are involved in transition of the cell from G1 to S phase

RUNX2 expression is regulated by estrogen signaling, and RUNX2 is implicated in breast cancer development and metastasis (reviewed in Wysokinski et al. 2014). Besides estrogen receptor alpha (ESR1) and estrogen-related receptor alpha (ERRA) (Kammerer et al. 2013), RUNX2 transcription is also regulated by TWIST1 (Yang, Yang et al. 2011), glucocorticoid receptor (NR3C1) (Zhang et al. 2012), NKX3-2 (BAPX1) (Tribioli and Lufkin 1999, Lengner et al. 2005), DLX5 (Robledo et al. 2002, Lee et al. 2005) and MSX2 (Lee et al. 2005). RUNX2 can autoregulate, by directly inhibiting its own transcription (Drissi et al. 2000). Several E3 ubiquitin ligases target RUNX2 for proteasome-mediated degradation: FBXW7a (Kumar et al. 2015), STUB1 (CHIP) (Li et al. 2008), SMURF1 (Zhao et al. 2003, Yang et al. 2014), WWP1 (Jones et al. 2006), and SKP2 (Thacker et al. 2016). Besides formation of RUNX2:CBFB heterodimers, transcriptional activity of RUNX2 is regulated by binding to a number of other transcription factors, for example SOX9 (Zhou et al. 2006, TWIST1 (Bialek et al. 2004) and RB1 (Thomas et al. 2001).

RUNX2 regulates expression of several genes implicated in cell migration during normal development and bone metastasis of breast cancer cells. RUNX2 stimulates transcription of the ITGA5 gene, encoding Integrin alpha 5 (Li et al. 2016) and the ITGBL1 gene, encoding Integrin beta like protein 1 (Li et al. 2015). RUNX2 mediated transcription of the MMP13 gene, encoding Colagenase 3 (Matrix metalloproteinase 13), is stimulated by AKT mediated phosphorylation of RUNX2 (Pande et al. 2013). RUNX2 is implicated in positive regulation of AKT signaling by stimulating expression of AKT-activating TORC2 complex components MTOR and RICTOR, which may contribute to survival of breast cancer cells (Tandon et al. 2014).

RUNX2 inhibits CDKN1A transcription, thus preventing CDKN1A-induced cell cycle arrest. Phosphorylation of RUNX2 by CDK4 in response to high glucose enhances RUNX2-mediated repression of the CDKN1A gene in endothelial cells (Pierce et al. 2012). In mice, Runx2-mediated repression of Cdkn1a may contribute to the development of acute myeloid leukemia (AML) (Kuo et al. 2009). RUNX2 can stimulate transcription of the LGALS3 gene, encoding Galectin-3 (Vladimirova et al. 2008, Zhang et al. 2009). Galectin 3 is expressed in myeloid progenitors and its levels increase during the maturation process (Le Marer 2000).

For a review of RUNX2 function, please refer to Long 2012 and Ito et al. 2015