**2.1 Phosphorylation of the p65 subunit of NF-ĸB and the role of its kinases in cancer**

Phosphorylation is a critical modification for NF-κB activation and plays an indispensable role in the regulation of its target genes. Moreover, as mentioned above, many of these target genes contribute to the hallmarks of cancer such as cellular proliferation, antiapoptosis as well as enhanced angiogenesis via vascular endothelial growth factor expression, among others [6]. Thus, understanding how phosphorylation of NF-κB contributes to these cancer phenotypes is a critical step in effectively limiting NF-κB activity [16]. Generally speaking, phosphorylation requires phosphorus which is supplied by the donor molecule adenosine tri-phosphate (ATP). Although several members of the NF-κB family of proteins are reported to be subjected to phosphorylation, p65 stands out as the most frequently modified subunit (**Table 1**). Furthermore, scientists have found that p65 can be phosphorylated by a variety of different kinases, some of which are themselves frequently overactive in cancer. For instance, p65 phosphorylation at serine 536 (S536) by IKKβ has been shown to be critical for TNFα-induced transformation of mouse epidermal cells [17]. Additional studies have also reported a role for p65 S536 phosphorylation in mediating expression of matrix metalloproteinase 1 (MMP-1) in lymphomas, wherein high MMP-1 expression correlated with lymphatic invasion and lymph node metastasis [18]. Another study with an immortalized prostate cell line, PNT1a, showed a role for phosphorylated S536 of p65 in cell motility and transformation [19].

As mentioned above, phosphorylation events on NF-κB are mediated by a variety of kinases. It is therefore unsurprising that the action of these kinases has been tightly regulated to maintain normal cellular function. However, deregulation


**Table 1.**

*List of known phosphorylation sites on the p65 subunit of NF-κB and their relationship to cancer.*

**51**

**chemoresistance**

*Phosphorylation of NF-κB in Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.83650*

of kinase activity can have detrimental downstream effects, which also involves the aberrant activation of NF-κB and its target genes to promote a cancer phenotype. Several kinases have been shown to have critical roles in the regulation of the p65 subunit of NF-κB. For example, glycogen synthase kinase 3 beta (GSK3β) and TRAF-associated NF-κB activator TBK1 ((TANK)-binding kinase 1) have been shown to be critical activators of NF-κB signaling [20, 21] by targeting p65 for phosphorylation on S536. This phosphorylation leads to enhanced NF-κB transactivation both *in vitro* and *in vivo* [22–24]. Another well-known kinase involved in modifying p65 is protein kinase A (PKAc). PKAc is typically activated following IκBα-degradation, leading to PKAc-mediated phosphorylation of p65 on S276 [25]. This phosphorylation event causes recruitment of histone acetyltransferases including cAMP response element-binding (CREB)-binding protein (CBP) and p300. The net effect is displacement of p50-histone deacetylase (HDAC)-1 complex from DNA, which increases p65 transactivation ability [26, 27]. Other kinases can also phosphorylate p65 at S276. These include mitogen- and stress-activated protein kinase-1 and 2 (MSK1, 2), proto-oncogene serine/threonine-protein kinase PIM-1 (PIM-1), ribosomal s6 kinase (RSK) p90, and protein kinase C α (PKCα) [28–31]. Moreover, casein kinase II (CK2), which phosphorylates p65 on S529, has been implicated in breast cancer [32, 33]. Another study demonstrated a role for p65 S276 phosphorylation by protein kinase A (PKA) in promoting a malignant phenotype in head and neck squamous cell carcinoma (HNSCC). Here, the authors found that S276 phosphorylation was prevalent in the nucleus of HNSCC samples but cytoplasmic in normal mucosa. Furthermore, this TNF-α-induced nuclear p65-S276 phosphorylation was significantly inhibited by the PKA inhibitor H-89, which in turn suppressed NF-κB activity, target gene expression, cell proliferation, and induced cell death via G1/S phase arrest [34]. Other p65-targeting kinases implicated in cancer include cell division protein kinase 6 (CDK6) and PIM-1, which phosphorylate p65 at S536 and S276, respectively [29, 35]. Both PIM1 and CDK6 have been shown to be overexpressed in a variety of cancers including hematological cancers, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, liver cancer, glioblastomas, medulloblastomas, colon cancer, and lung cancers [36–39]. However, their exact roles in regulating p65 phosphorylation in these cancers are yet to be understood. In gastric cancer cells, Aurora Kinase A (AURKA) was also shown to phosphorylate S536 in *in vivo* and *in vitro* models [40] whereby overexpression of AURKA induced a significant increase in NF-κB p65 and phospho-p65 (S536) protein levels. Interestingly, protein kinase C δ (PKCδ), a member of novel PKC isoforms, has also been implicated in a number of cancers including breast, pancreatic, prostate, and melanoma tumor cells but has been shown to regulate p65 transactivation in a phosphorylation-independent manner [41]. Our laboratory has also recently reported a novel phosphorylation site on S316 of p65, a modification mediated by the kinase CKII [41]. We showed that S316 phosphorylation was necessary for NF-κB activation and target gene expression. Collectively, these examples indicate the importance and sophistication of p65 phosphorylation and their corresponding kinases in regulating NF-κB signaling in the context of cancer.

**2.2 Importance and effects of phosphorylation of p65 in modulating** 

upon knockdown of p65 [42]. These and other accounts of NF-κB-mediated

Several studies have indicated a role for NF-κB hyperactivity in the development of resistance to chemotherapeutics via downregulation of antisurvival and upregulation of prosurvival target genes and pathways [42–44]. In one study for example, gemcitabine-resistant pancreatic cancer cells were rendered sensitive to gemcitabine

### *Phosphorylation of NF-κB in Cancer DOI: http://dx.doi.org/10.5772/intechopen.83650*

*Adenosine Triphosphate in Health and Disease*

**in cancer**

**Known phosphorylation** 

**sites of p65**

**2. Role of phosphorylation of NF-κB in cancer**

**2.1 Phosphorylation of the p65 subunit of NF-ĸB and the role of its kinases** 

phosphorylated S536 of p65 in cell motility and transformation [19].

As mentioned above, phosphorylation events on NF-κB are mediated by a variety of kinases. It is therefore unsurprising that the action of these kinases has been tightly regulated to maintain normal cellular function. However, deregulation

S205 No cancers currently known HEK 293 cells [104]

S529 Breast cancer HeLa cells [33] S536 Bone cancer HeLa and BC-3 cells [35]

S281 No cancers currently known MEF cells [106]

T435 No cancers currently known SiHa cells [108] S468 No cancers currently known HeLa cells [109]

S535 No cancers currently known HeLa cells [111] S316 No cancers currently known HEK 293 cells [41] S547 No cancers currently known HEK 293 cells [112]

T254 Breast cancer BT20 and MCF-7

S311 No cancers currently known HEK 293 and MEF

T505 No cancers currently known NARF2 and Hs68

*List of known phosphorylation sites on the p65 subunit of NF-κB and their relationship to cancer.*

S276 Head and neck cancers, breast cancer

**Cancers involved Cell line discovered References**

cells

cells

cells

HNSCC cells [34]

[105]

[107]

[110]

Phosphorylation is a critical modification for NF-κB activation and plays an indispensable role in the regulation of its target genes. Moreover, as mentioned above, many of these target genes contribute to the hallmarks of cancer such as cellular proliferation, antiapoptosis as well as enhanced angiogenesis via vascular endothelial growth factor expression, among others [6]. Thus, understanding how phosphorylation of NF-κB contributes to these cancer phenotypes is a critical step in effectively limiting NF-κB activity [16]. Generally speaking, phosphorylation requires phosphorus which is supplied by the donor molecule adenosine tri-phosphate (ATP). Although several members of the NF-κB family of proteins are reported to be subjected to phosphorylation, p65 stands out as the most frequently modified subunit (**Table 1**). Furthermore, scientists have found that p65 can be phosphorylated by a variety of different kinases, some of which are themselves frequently overactive in cancer. For instance, p65 phosphorylation at serine 536 (S536) by IKKβ has been shown to be critical for TNFα-induced transformation of mouse epidermal cells [17]. Additional studies have also reported a role for p65 S536 phosphorylation in mediating expression of matrix metalloproteinase 1 (MMP-1) in lymphomas, wherein high MMP-1 expression correlated with lymphatic invasion and lymph node metastasis [18]. Another study with an immortalized prostate cell line, PNT1a, showed a role for

**50**

**Table 1.**

of kinase activity can have detrimental downstream effects, which also involves the aberrant activation of NF-κB and its target genes to promote a cancer phenotype. Several kinases have been shown to have critical roles in the regulation of the p65 subunit of NF-κB. For example, glycogen synthase kinase 3 beta (GSK3β) and TRAF-associated NF-κB activator TBK1 ((TANK)-binding kinase 1) have been shown to be critical activators of NF-κB signaling [20, 21] by targeting p65 for phosphorylation on S536. This phosphorylation leads to enhanced NF-κB transactivation both *in vitro* and *in vivo* [22–24]. Another well-known kinase involved in modifying p65 is protein kinase A (PKAc). PKAc is typically activated following IκBα-degradation, leading to PKAc-mediated phosphorylation of p65 on S276 [25]. This phosphorylation event causes recruitment of histone acetyltransferases including cAMP response element-binding (CREB)-binding protein (CBP) and p300. The net effect is displacement of p50-histone deacetylase (HDAC)-1 complex from DNA, which increases p65 transactivation ability [26, 27]. Other kinases can also phosphorylate p65 at S276. These include mitogen- and stress-activated protein kinase-1 and 2 (MSK1, 2), proto-oncogene serine/threonine-protein kinase PIM-1 (PIM-1), ribosomal s6 kinase (RSK) p90, and protein kinase C α (PKCα) [28–31]. Moreover, casein kinase II (CK2), which phosphorylates p65 on S529, has been implicated in breast cancer [32, 33]. Another study demonstrated a role for p65 S276 phosphorylation by protein kinase A (PKA) in promoting a malignant phenotype in head and neck squamous cell carcinoma (HNSCC). Here, the authors found that S276 phosphorylation was prevalent in the nucleus of HNSCC samples but cytoplasmic in normal mucosa. Furthermore, this TNF-α-induced nuclear p65-S276 phosphorylation was significantly inhibited by the PKA inhibitor H-89, which in turn suppressed NF-κB activity, target gene expression, cell proliferation, and induced cell death via G1/S phase arrest [34]. Other p65-targeting kinases implicated in cancer include cell division protein kinase 6 (CDK6) and PIM-1, which phosphorylate p65 at S536 and S276, respectively [29, 35]. Both PIM1 and CDK6 have been shown to be overexpressed in a variety of cancers including hematological cancers, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, liver cancer, glioblastomas, medulloblastomas, colon cancer, and lung cancers [36–39]. However, their exact roles in regulating p65 phosphorylation in these cancers are yet to be understood. In gastric cancer cells, Aurora Kinase A (AURKA) was also shown to phosphorylate S536 in *in vivo* and *in vitro* models [40] whereby overexpression of AURKA induced a significant increase in NF-κB p65 and phospho-p65 (S536) protein levels. Interestingly, protein kinase C δ (PKCδ), a member of novel PKC isoforms, has also been implicated in a number of cancers including breast, pancreatic, prostate, and melanoma tumor cells but has been shown to regulate p65 transactivation in a phosphorylation-independent manner [41]. Our laboratory has also recently reported a novel phosphorylation site on S316 of p65, a modification mediated by the kinase CKII [41]. We showed that S316 phosphorylation was necessary for NF-κB activation and target gene expression. Collectively, these examples indicate the importance and sophistication of p65 phosphorylation and their corresponding kinases in regulating NF-κB signaling in the context of cancer.
