Author: Janice Rateshwar
Peer Reviewer: Sammer Marzouk
Professional Reviewer: Sidra Tul Muntaha
Introduction
Tumor suppressor proteins regulate normal cell proliferation and survival (Fabbro & Henderson, 2003). Namely, the tumor suppressor protein, p21, is known for its ability to mediate the cell cycle by inhibiting several cyclin and Cyclin-Dependent Kinase (cyclin – CDK) complexes. p21 also has a role in DNA repair through its interaction with proliferating cell nuclear antigen, PCNA (Sherr & Roberts, 1999; Abbas & Dutta, 2009; Young Lee, Suk Kim, Young Kim, & Sohn, 2009; Georgakilas, Martin, & Bonner, 2017). Furthermore, p21 has exhibited a crucial role in determining the fate of cancer progression. p21 has shown a correlation with the prognosis colorectal cancer, head and neck cancer, and small-cell lung cancer (Piccolo & Crispi, 2012). However, p21 undergoes induction through various environmental genotoxic agents and has multiple effects on cell survival. Although p21 is known to be a tumor suppressor protein, in some cases it also displays oncogenic behavior. When expressing anticancer properties, p21 promotes cell death, inhibits cell proliferation, and induces senescence on cells containing damaged DNA. On the other hand, when expressing cancerous properties, p21 inhibits cell death, promotes cell proliferation, and hinders senescence on cells containing damaged DNA. There have been many factors which impact this dual behavior such as, its role in cyclin-CDK complex assembly, its function in a p53 deficient and sufficient environment, and its method of induction (Sherr & Roberts, 1999; Gartel, 2006; Abbas & Dutta, 2009; Georgakilas et al., 2017). Recent research has found the subcellular localization of p21 to be the predominant factor influencing the dual properties of p21 as both pro-cancerous and anti-cancerous (Xia et al., 2004; Arai, Nomura, Fukuchi, & Gomi, 2006; Piccolo & Crispi, 2012). Knowing that p21 exhibits an oncogenic effect on multiple cancer treatments, this research examined methods through which subcellular localization can be altered to increase collaboration between p21 and current cancer treatments. Nuclear accumulation of p21 through various mechanisms leads to increased effectiveness of cancer treatments. Future research can be conducted to discover additional methods through which nuclear accumulation of p21 can assist cancer treatments.
Subcellular localization on the cancerous/anti-cancerous properties of p21
The subcellular location of p21 is crucial for determining its role in cancer. Nuclear p21 exhibits tumor suppressor activity, as shown by its negative regulation of the cell cycle through interaction with CDK/Cyclin complexes. On the other hand, Cytoplasmic p21 displays oncogenic behavior as shown by its inhibition of apoptosis and induction of cell proliferation (Piccolo & Crispi, 2012). The multiple roles of p21 in cancer can be better understood through analyzing the behavior of nuclear p21 in comparison to that of cytoplasmic p21.
Nuclear p21
Nuclear p21 has significant anticancer properties. When localized in the nucleus, p21 inhibits cyclin-CDK at the growth one or growth two/mitosis checkpoints and therefore promotes cell cycle arrest. One of the significant properties of nuclear p21 is its ability to increase apoptosis (Ritt et al., 2000). It also limits the proliferation of stem cells and induces senescence. Wild-type p21 is located in the nucleus and is affected by the nuclear localization signal (NLS) at the C-terminal domain. The nuclear localization regulates nuclear translocation of p21 through identifying importins, which modulate the protein’s passage through nuclear pores (Georgakilas et al., 2017). Additionally, the bipartite NLS, either located in the RKRR or RKRR qts mtdf-yh sKRR amino acid sequence, is needed for the nuclear translocation of p21 (Rodr?guez-Vilarrupla et al., 2002). On the N- terminal domain of p21, there are three nuclear localization signals. In order for the elimination of nuclear translocation and all three must have mutations. (Singh, Song, Yang, & Kumar, 2005). In short, the nuclear localization signal is vital for nuclear accumulation of p21.
Cytoplasmic p21
On the contrary, the cytoplasmic expression of p21 is correlated with more aggressive tumors and poorer cancer prognosis. When localized in the cytoplasm, p21 exhibits cancerous properties. Cytoplasmic p21 promotes cell survival by inhibiting cytoplasmically localized apoptosis-related proteins and promoting cellular proliferation through both the mitigation of CDK2 and PCNA inhibition and the assembly of the D-type cyclins (D1, D2, and D3) with CDK4 and CDK6. For instance, only in the presence of p21-cyclin D complexes, does p21 promote oligodendrogliomas formation. However, the leading cause behind the cytoplasmic localization of p21 is phosphorylation at Ser130 and Thr145 (Abbas and Dutta, 2009; Child & Mann, 2006). Protein kinase B, Akt, is a serine/threonine protein kinase which promotes phosphorylation and inhibits the apoptotic properties of p21 (Jänicke, Sohn, Essmann, & Schulze-Osthoff, 2007). In particular, Akt phosphorylates a threonine residue at the C-terminal NLS of p21. Although Akt activation does not affect the total levels of p21, it does alter its subcellular distribution: when the phosphorylation of the NLS is reduced, the protein is predominantly nuclear, but when p21 phosphorylated, it is cytoplasmic (Schmeichel, 2001). Nuclear export is signaled through Akt-mediated phosphorylation of HER2/neu and/or phosphorylation by protein kinase C, PKC (Turner, Dawson, & Sullivan, 2012; Schmeichel, 2001). Interestingly, full-length cytoplasmic p21 does not have anti-apoptotic properties (Arai et al., 2006). Moreover, transfecting hepatic carcinoma cells with a mutated NLS showed increased proliferation of anti-apoptotic behavior (Piccolo & Crispi, 2012). In clinical research, more cytoplasmic accumulation and phosphorylation of p21 appears to be correlated with the overexpression of HER2/neu and Phospho-Akt and a lower survival rate of a breast cancer patient (Xia et al., 2004). In summary, cytoplasmic accumulation of p21 through phosphorylation at Ser130 and Thr145 influences p21 to act in an oncogenic manner.
Targeting Nuclear Accumulation
Given the importance of nuclear accumulation in battling cancer, it is vital to examine pathways and molecules which assist the nucleic buildup of p21, for example, downregulation of BRCA2 and CDKN1A Interacting Protein, BCCIP, reduces nuclear accumulation and promotes cytoplasmic localization (Fan, Wray, Meng, & Shen, 2009). Interestingly, the nucleolus may affect the nuclear export of p21. Intranucleaolar bodies during cell stress contain small ubiquitin-like modifiers or SUMOs which regulate cyto-nucleic transport. SUMOylation regulates the localization of p21 by nuclear pores through SENP2, a deSUMOylation enzyme. A p21 – SUMO complex localized in the nucleolus can inhibit disruption of nuclear export caused by DNA damage (Brun et al., 2017). Moreover, it is necessary for oncogenic protein Brap2 to bind to the NLS of BRCA1 for cytoplasmic localization of p21 to occur. Brap2-targeting siRNA interferes with the ability of Brap2 to bind to the NLS of BRCA1 and therefore decreases cytoplasmic expression and apoptosis (Asada et al., 2004). All these methods are viable options for developing more effective cancer treatments in the future.
Potential Positive Effect on Cancer Treatments
p21 has a dual impact on cancer treatments as well. In some cases, p21 increases the susceptibility of cancer cells to treatment and reduces the side effects of treatments, while in other cases, it does the opposite. (Gartel, 2006; Georgakilas et al., 2017; Piccolo & Crispi, 2012). Nuclear accumulation promotes collaboration between p21 and cancer treatments. For example, cargo proteins, transport receptors, RAN regulators, and NPC effectively influence the subcellular localization of proteins in diseased states. Future research can examine the effect of these molecules on the relationship between cancer treatments and p21. However, at the moment, only CRM1 and importin-?/ß are being actively targeted to interfere with the subcellular localization of p21 (Hill, Cautain, Pedro, & Link, 2014).
CRM1
In particular, low-toxicity small-molecule CRM1 inhibitors have recently started being tested in cancer cells and are correlated to decreased nuclear export and drug resistance. One specific type of small molecule inhibitors of CRM1, selective inhibitors of nuclear export or SINE which inhibit CRM1 are now even orally available . The positive results from CRM1 and importin-?/ß targeted treatment are vital in opening future windows in stabilizing the dual-tumor suppressor proteins (Turner et al., 2012; Hill et al., 2014; Ohkoshi, Yano, & Matsuda, 2015).
Antineoplastic Drugs
Antineoplastic drugs are medications used to treat cancer. The antineoplastic drug, cisplatin, is often the primary treatment for several cancers. However, throughout the treatment, patients may develop resistance to the drug. Therefore, its success is limited due to dose-limiting toxicity (Basu & Krishnamurthy, 2010). Interestingly, it is found that cancer cells with cytoplasmic p21 tend to resist cisplatin-induced apoptosis (Ohkoshi et al., 2015). However, various mechanisms can alter the subcellular localization of p21 to make it more compatible with cisplatin-induced cancer treatment. For example, epigallocatechin gallate, an active component of green tea, and sulforaphane, an active element in broccoli can be fused into a combined treatment which upregulates p21 more than their individual treatments would and allows for the cisplatin treatment to be more effective in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells (Chen, Landen, Li, Alvarez, & Tollefsbol, 2013). Likewise, p21 increases the susceptibility of osteosarcoma cells to cisplatin-induced-cytotoxicity through activating the caspase-3 cascade and increasing Bax/Bcl-2 ratio (Ding et al., 2013). Lastly, p21-mediated apoptosis in cell lines of malignant mesothelioma based on the piroxicam and cisplatin combined treatment promotes apoptosis and nuclear accumulation of p21. Through microarray data and qPCR, scientists hypothesized p21 helps piroxicam functionality and makes cancer cells susceptive to cisplatin treatment. When p21 was silenced by siRNA, there was a decrease in piroxicam-cisplatin induced apoptosis (Baldi et al., 2011). The well-known cancer medication, calmodulin, also inhibits the phosphorylation of wild-type 21 through PKC (Protein Kinase C) activation at Ser153, and thus, prevents its nuclear export (Rodríguez-Vilarrupla et al., 2005; Child & Mann, 2006).
Negative Effect On Cancer Treatments
On the contrary, the newly synthesized chemical compound, Rosiglitazone Metformin, can upregulate p21 by activating the AMPK/p21 pathway which promotes cytoplasmic localization of p21 and thus promotes cellular proliferation (Liu, Hu, Shan, Chen, & Tang, 2019). Additionally, downregulation of p21 in irinotecan and flavopiridol treatments increases the success rate. However, the subcellular localization of p21 is not specified in these researches, and future research for the subcellular localization of p21 in these treatments can help improve their success rate. For example, Doxorubicin is a chemotherapy drug which can cause heart failure in the long run. One study found that Doxorubicin treatment increased p21 expression is correlated with increased mitochondrial fission and decreased apoptosis in cardiomyocytes; however, this study did not specify the subcellular localization of p21 (Wan et al., 2019). Another study found that cells treated with adriamycin, the brand name for Doxorubicin, contained nucleolar p21 and promoted nuclear accumulation. These results propose a potential anticancer role of p21 in Doxorubicin treatment (Abella et al., 2010). Furthermore, cytoplasmic p21 promotes resistance of the chemotherapy drug, 5-Fluorouracil in colorectal cancer cells by shuttling p-Chk2 out of the nucleus (Maiuthed et al., 2018). By identifying the subcellular localization of p21 during these cancer treatments, scientists can improve their prediction regarding the function of p21.
Conclusion
The subcellular location of p21 is exceptionally significant in determining its role in cancer. Nuclear p21 has several anticancer properties, while cytoplasmic p21 actively exhibits oncogenic behavior. Targeting nuclear accumulation of p21 can be the key to strengthening the relationship between p21 and current cancer treatments.
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