Restoration of Tumor Suppression to Cancer Carrying p53 Mutations

Abstract

Missense mutations of tumor suppressor genes enable cancerous cells generating variable mutant proteins and promote malignant development. These mutant proteins lose the original functions in suppressing tumorous cells but also commit oncogenic activities to tumor progression. Targeting mutants of the p53 tumor suppressor merges a specific approach for cancer treatments. This chapter will highlight the progress from our group and those of others in this filed. We will introduce new concepts and molecular mechanisms underlying the expression of mutant proteins and cancer resistance to conventional treatments. Furthermore, we will introduce the potential agents holding great promises in preclinic studies for cancer treatments.

1. Introduction

The tumor suppressor p53 plays a pivotal role in maintaining genome integrity and protecting the cells from neoplastic transformation. Apart from its well-known function in cell-cycle arrest, DNA repair, apoptosis, and senescence, recent reports show that p53 also regulates the stemness of cells and immune response against tumorigenesis and tumor progression [1, 2, 3]. p53 maintains homeostasis between self-renewal and differentiation and prevents either dedifferentiation or reprogramming of somatic cells to cancer stem-like cells [2]. By modulating transcription of genes, those proteins are involved in regulating immune recognition and response, p53 conserves immune surveillance and destruction against cancerous cells.

TP53 gene is frequently mutated in almost of all types of human cancer. The majority of its genetic alterations are missense mutations occurred in the DNA-binding domain (DBD) [4, 5]. p53 hotspot mutations, which are frequently detected missense mutants and located in the DBD, can be categorized into “contact” mutants (R248, R273) and conformational mutants (R175H, G245, R249, and R282) [6, 7]. These mutants not only lose the canonical tumor-suppressive functions of the wild-type p53 (wt-p53) but also commit any gain-of-function (GOF) of mutant p53 (mut-p53) that favors cancerous cell survival and promotes tumor progression [7, 8, 9]. mut-p53 proteins primarily execute as transcription factor, regulate the expression of several genes and non-coding RNAs, and confer oncogenic properties, including sustained proliferation, resistance to cell death, replicative immortality, and evading immune destruction [10]. In recent years, novel functions of mut-p53 in promoting dedifferentiation of somatic cells to cancer stem cells (CSCs) and in avoiding antitumor immunity have gathered considerable attention. Cancer cells that carry mut-p53 not only withstand the genomic instability but also adore oncogenic signals to malignant transformation. The correlation of GOF mut-p53 to cancer stemness was realized in the undifferentiated thyroid carcinoma [11] and a poor prognosis of patients with cancers [12]. This was further supported by GOF mut-p53 endorsed tumorigenicity of embryonic stem cells (ESCs) and these mut-p53 dependent ESCs share the gene signature with undifferentiated tumors carrying mut-p53 [2, 13]. GOF mut-p53 can alter the immunogenicity of cancer cells and further confine immune escape [3, 14].

Since p53 missense mutation is the most come genetic alterations in cancers, targeting these GOF mut-p53 holds a great promise for developing selective and effective treatments against cancer. In this chapter, we will briefly review the progress in GOF mupt-p53 and cancer progression and discuss various mechanisms driving alteration of cellular plasticity and the immune response upon mut-p53 and the efforts to delineate novel ways to specifically target CSCs residing in mut-p53 tumors and enhance antitumor immunity.

2. p53 mutations and cancer

2.1 p53 transactivates and regulates the expression of p53-target genes to suppress tumor

Tumor suppressor p53 acts as a central regulator in a myriad of cellular signaling pathways that control the cell cycle and maintain the integrity of the human genome [15]. p53 functions primarily as a transcription factor for modulating the expression of approximately 350 p53-target or responsive genes [5, 16, 17]. p53 protein is biologically active as a homotetramer (4 × 393 amino acid residues) and its domain structure contains a transactivation domain (TA), a proline-rich region (PR), DNA-binding domain (DBD), oligomerization domain (OD), and regulatory domain (Reg), sequentially located from amino terminus to carboxyl terminus (Figure 1a,c). The core domain, DBD (residues 94–292) forms a structure, including a central β-sandwich scaffold and additional elements, providing the DNA-binding surface [18, 19]. This p53 core domain has a low thermodynamic stability and it can rapidly unfold at body temperature (t_1/2, 9 min) [20, 21]. This low stability is linked with the structural plasticity required to facilitate p53 binding to DNA and other proteins, and may have implications with the susceptibility to mutations frequently detected in cancers [5, 22].

Figure 1.

p53 mutation and cancer development. a, p53 mutation turns tumor suppressor into oncogenic factors. Wild-type p53 protein (wt) forms a tetrameric transcription factor and suppresses tumorigenesis and tumor progression via apoptosis, cell differentiation, and immune response. Wt, wild-type of p53; Mut, mutant p53. b, hotspot p53 mutants. Among p53 mutations, all hotspot missense mutations are more commonly detected than others in cancer cases and all are located in the DNA-binding domain (DBD). Further, missense proteins caused by adenosine in each hotspot mutated codon are more common than others. c, p53 domain structure. TAD, transactivation domain; PRR, proline-rich region; DBD, DNA-binding domain; OD, oligomerization domain; Reg, regulatory domain.

2.2 Hotspot mut-p53 in the DBD display oncogenic function

GOF Tumor suppressive function of p53 is inactivated by genetic mutations in approximately 42% of cancers, and its apoptotic pathways are impaired in those of other cancers. Mut-p53 proteins in cancer cells, either homotetramer or heterotetramer, not only lose the tumor suppressor function of wt-p53 but also endow the gain-of-function (GOF) that displays oncogenic activity, contributing to cancer progression (Figure 1a) [23]. Mutations in the TAD lead to truncated p53 that can activate apoptotic target genes [24]. However, most mutations occur in DBD and lead to functional inactivation. A majority of TP53 mutations (~80%) are detected in the exons 5–8 that encode the DBD of p53 protein (Figure 1c) [25]. The main mutant types of TP53 include missense mutations (~73%), frameshift mutations (~11%), non-sense mutations (~7%), silencing mutations (~5%), and splice mutations (~2%) [6, 26]. Missense mutations of TP53 result in single amino acid substitutes, which can present GOF activity during tumorigenesis, such as p53 R175H and p53 R273H that promote tumor invasion and migration [27, 28, 29]. Splice mutations generate alternative proteins that can also advance tumor development [30]. Among p53 mutants, the most common mutation sites that are called hotspot mutations in cancer cases occur at the codons of R175, G245, R248, R249, R273, and R282 (Figure 1b). Based on the COSMIC Database (https://cancer.sanger.ac.uk/signatures/), it has been found that the most substitution mutations among mut-p53 are G to A transitions (G > A, 29.05%), followed by C to T transitions (C > T, 25.90%) [31]. The transited “A” from others (C > A, G > A, T > A) accounts for 34.82% of all p53 missense mutants. Interestingly, G > A (~15% of all) is major transition for R175, G245, R248 and R273 hotspot mutations (Table 1 and Figure 1b). mut-p53 is usually divided into two categories. One category is DNA contact mutations (contact mut-p53), which occur in the amino acids in contact with DNA and cause inability of mut-p53 protein binding to DNA, such as p53 R273H and p53 R248Q. Another category is conformational mutations (conformational mut-p53), which occur in amino acids that maintain the structure and can result in unfolded proteins, such as p53 R175H, p53 Y220C, and p53 R249S [32].

Mutation	Overall Frequency	Wild-Type Codon	Mutant Codon	Class of Activation	GOF
G > A (~14.7%)
R175H	4.6%	CGC	CAC	Conformation	GOF
R248Q	3.5%	CGG	CAC/CAA	DNA contact	GOF
R273H	3.1%	CGT	CAT	DNA contact	GOF
R245S	2.8%	GGC	AGC	Conformation
G245D	0.68%	GGC	GAC	Conformation
C > T or G > T
G248W	2.8%	CGG	TGG	DNA contact	GOF
R273C	2.7%	CGT	TGT	DNA contact
R282W	2.4%	CGG	TGG	DNA contact	GOF
R249S	1.8%	AGG	AGT	Conformation

Table 1.

Hotspot p53 mutations and their codon transitions in human cancer.

G > A, guanine transited to adenine; C > T, cytosine transited to thymine; G > T, guanine transited thymine.

2.3 Mut-p53 is a specific target for cancer treatments

mut-p53 proteins exert oncogenic GOF mainly via altered transcriptional mechanisms. wt-p53 protein recognizes and binds to DNA response elements, then recruits transcription factors, histone acetyltransferases, chromatin remodeling complexes, to form the pre-initiation complex for transcription [31, 33]. It has been reported that mut-p53 cannot bind to the response element for wt-p53, but exerts its GOF activity via different mechanisms. For instance, mut-p53 can bind to diverse transcription factors and cofactors (NF-Y, p73, NRF2, Ets-1), thus altering transcription of target genes [33]. In cancer cells upon DNA damage, mut-p53/NF-Y complex recruits p300 and then binds to NF-Y target promoters, leading to histone acetylation and overexpression of NF-Y target genes permitting tumor progression [34]. In some cases, mut-p53 (G245S) can directly bind to DNA with some specific structures, such as nuclear matrix attachment regions (MARs), and regulate transcriptions [35]. mut-p53 also can interact with other cellular proteins, thereby altering the functions of these proteins. In the knock-in mice, mut-p53 (R270H) antagonized the p63/p73-regulated transcription for tumor suppression via Notch1 pathway, and then promoted tumorigenesis and tumor progression [36]. It is noted that altered cellular localization of particular mut-p53 also contributes to its GOF. p53 E258K, R273H, and R273L mutants, which were located in the cytoplasm, could inhibit autophagy in colon cancer cells [37].

mut-p53 spectrum differs between tumors and is associated with poor prognosis in cancer patients. Frequency of mut-p53 in tumor tissue samples from 10,000 cancer patients is 42% (https://www.cbioportal.org/). However, the frequency is significantly higher in small cell lung cancer (89%) and in colorectal cancer (73%). Strikingly, mut-p53 is highly associated with a poor prognosis in different types of cancers observed. The cBioportal for Cancer Genomics Database revealed that mut-p53 expression is negatively correlated with overall survival of patients with breast cancer, pancreatic cancer, hepatobiliary cancer, bone cancer, non-small cell lung cancer, and thyroid cancer [31]. For example, in breast cancer cases, although mut-p53 is in 30–35% all cases, mut-p53 is often detected in ~80% of triple-negative (TN) breast cancer, which always are poorer in prognosis than any other subtypes of breast cancer [38].

3. p53 mutants promote cancer stemness

The major oncogenic properties of GOF mut-p53, including enhanced metastasis, chemoresistance, and angiogenesis are integral to cancer stem cell (CSC). Indeed, recent studies indicate that GOF mut-p53 (R175H, R248W, R273H) promotes cancer stemness.

3.1 p53 maintains the balance of self-renewal and differentiation for homeostasis

By controlling the proliferation, differentiation, and apoptosis, p53 plays a significant role in ensuring genomic integrity of normal stem cells. Apart from its classical function, p53 also maintains tissue hemostasis between self-renewal and differentiation [1, 39]. p53 acts as a barrier to somatic cells counteracting the reprogramming process [39]. Human somatic cell can be reprogrammed to induced pluripotent stem (iPS) cells by induction of reprogramming factors (Oct4, SOX2, KLF4, and c-MYC) that are highly expressed in ESCs and regulate the signaling required for pluripotency (Figure 2) [40]. Silencing p53 expression with siRNA in adult fibroblasts can enhance the efficacy of generating iPS cells up to 110-fold [41]. Conversely, reduction of p53 signaling, by deleting or knocking down p53 or its target gene p21, increases reprogramming efficiency [42]. Moreover, p53 may upregulate miR-199a-3p expression so as in turn impose G1 arrest to decrease reprogramming efficacy [43]. mut-p53 plays a critical role in driving CSC phenotype [29, 44, 45]. GOF mut-p53 proteins lack the DNA-binding ability to p53-target genes, instead, they can piggyback on other transcription factors to regulating expression of a large number of genes and non-coding RNAs for malignant stemness.

Figure 2.

p53 mutants determinate cancer Stemness. p53 regulates the balance of cell proliferation and differentiations of cell population for homeostasis. Mut-p53 can result in either the reprograming of adult stem cells or dedifferentiating cancer cells to form cancer stem cells. Further, Mut-p53 sustains cancer stemness via enhancing the expression of genes involved in signaling pathways of NOTCH, WNT, hedgehog, BMI, and PTEN, thus promoting tumor metastasis, drug resistance, and even recurrence. Wt, wild-type; CSC, cancer stem cell; miR, microRNA; EMT, epithelial-mesenchymal transition.

3.2 GOF mut-p53 increases cancer stemness

CSC or cancer stem-like cell excuses as seed for tumorigeneses, and further promotes cancer progression as well as resistance to therapies. wt-p53 protein regulates the quantity and quality of adult stem cells to ensure normal tissue development with less tumorigenic risk. However, mut-p53 proteins, which inactivate p53 signaling and display GOF, can disrupt this balance, thereby promoting pluripotency and reprogramming somatic cells, including adult stem cells for initiating tumor [2]. Recent studies indicated that prevalent mut-p53 (R175H, R273, R248W) boosts the stemness properties of cancer cells (Figure 2) [29, 46, 47, 48]. Either “contact” or “conformation” mut-p53 in DBD execute GOF properties in favor cell survival and promoting tumor progression [8]. The potential role of mut-p53 played in CSC formation was realized from the correlation of undifferentiated tumors to mut-p53 in thyroid cancers [11]. Undifferentiated tumors of breast and brain expressed the same gene signature as embryonic stem cells (ESCs) [49]. Further, it has been recognized that the novel property of mut-p53 not only enhances the reprogramming efficiency of somatic cells but also promotes malignant potentials of mouse embryonic fibroblasts (MEFs) [13]. Introducing pluripotent factors (Oct4, Sox2, c-Myc, and KLf4) into adult differentiated cells can reprogram them to their embryonic state or result in dedifferentiation.

3.3 Mut-p53 promotes cell reprogramming/de-differentiation to enrich CSCs

GOF mut-p53 (R172H, corresponding to the R175H in human) enhanced the efficiency of the reprogramming process compared to p53 deficiency in MEFs [13]. Importantly, mut-p53 induced alterations in the reprogrammed cells to malignancy [13]. Although p53-knockout MEFs maintained the pluripotent capacity in vivo after the reprogram with Oct4 and Sox2, the mut-p53 cells displayed malignant pluripotency and generated tumors (Figure 2) [13]. While reprogrammed cells with p53-deficiency formed differentiated teratomas, mut-p53 reprogrammed MEFs formed undifferentiated malignant tumors [13]. This clearly indicates that GOF mut-p53 not only eliminates cancerous cells by apoptosis but also advances malignant pluripotency. A set of miRNAs has been identified, whose expression is altered (increased miR-15b, decreased miR-155) in a p53-dependent manner during transition of MEFs to induced pluripotent stem cells and these miRNAs may regulate the mut-p53 (R172H)-driven stemness [50]. Our group reported that introducing mut-p53 (R273H) via CRISPR/Cas9 editing into colorectal cancer SW48 cells markedly increased CSC population [29]. These mut-p53 cells that expressed low levels of p21 and PUMA while expressing high levels of Klf4, Oct4, c-Myc, TGF-β, and Zeb1, together promoted cancer cells to EMT and enhanced tumorigenesis in vitro and in vivo [29]. These indicate that mut-p53 (R273H) can reprogram or dedifferentiate cells to enhance cancer stemness in Colorectal cancer (Figure 2). Other reports showed that colorectal cancer SW480 cells (mut-p53 R273, P309S) harbored an increased population of CD44, Lgr5, and ALDH positive CSCs, as mut-p53 transcriptionally upregulates these CSC markers to promote CSC population [45]. Other works revealed that GOF mut-p53 (R175H, R273H) promotes cancer stemness through PI3K/AKT2-mediated integrin and growth factor signaling in cancer cells of glioblastoma and breast cancer [44]. AKT2 can enhance the phosphorylation of WASP interacting protein (WIP) and stabilize YAP/TAZ, thus supporting SCS survival and phenotypic maintenance (Figure 2) [44]. Mut-p53 can also induce the nuclear accumulation of YAP/TAZ and the activity via interacting with SREBP transcription and increased geranylgeranyl pyrophosphate from mevalonate biosynthesis for promoting self-renewal of breast cancer cells [51].

CSCs also can be derived from cancer cells. There are two CSC populations existed in tumors: the intrinsic CSCs that are inherently present in the tumor and the induced CSCs that arise from differentiated tumor cells as a consequence of EMT signaling [52]. During cancer development, a small numbers of aggressive cancer cells possessing cellular plasticity undergo reversible transformations, including epithelial to mesenchymal transition as well as mesenchymal to epithelial transition, and migrate to other tissues or organs to form metastasis [53]. Cancerous EMT is different from the embryonic one and requires CSCs being able to intravasate/extravasate and colonize at distant sites. EMT of cancer cells can be triggered by many extracellular signals, including transforming growth factor b (TGF-β), bone morphogenetic proteins (BMPs), Wnt, Notch, epidermal growth factor, fibroblast growth factor, and many others [54]. These signals, via modulating certain transcription factors (TFs) (such as Snail, Twist, Zeb, and others) independently or in combination, suppress epithelial phenotype, and further enhance mesenchymal traits, including enhanced migratory capacity, invasiveness, resistance to apoptosis, and production of extracellular matrix ECM components [55, 56]. These “migratory cells”, after EMT, have the ability to form secondary tumors and differentiate into non-stem cancer cells, which are the very traits of self-renewal, and are increased resistance to chemotherapy and highly metastatic [52, 57].

One of the major GOF properties of mutant p53 is invasion and metastasis. Mut-p53-induced EMT triggers stemness properties in cancer cells and enriches CSCs in tumors under treatments [29, 58]. wt-p53 promotes epithelial differentiation through transcriptional activation of miR-200c [59], which inhibits the translation of EMT activator Zeb1/Zeb2 and then represses the expression of self-renewal factors like Bmil and possibly Klf4 and Sox2 [60, 61]. Conversely, loss of p53 in mammary epithelial cells reduces the expression of miR-200c and promotes EMT and stemness properties, thus generating high-grade breast tumors [59]. These findings were corroborated that loss of p53 increased levels of stemness regulators (Bmi1, Klf4, Vimentin) and EMT inducers (Snail, Twist, Zeb1, and Zeb2) in pancreatic acinar cells [62, 63]. p53 has also been implicated in suppressing EMT and the stemness of PC-3 prostate cancer cells by miR-145 [64]. PC3 cancer cells carrying wt-p53 expressed high levels of epithelial marker E-cadherin, while presented reduced levels of mesenchymal markers (fibronectin, vimentin, N-cadherin, and Zeb2) as well as CSC markers (CD44, Oct4, c-Myc, Klf4). Inhibition of miR-145 promoted EMT, as increased mesenchymal markers and CSC markers in those cells [64]. These indicate that wt-53 plays a crucial role in maintaining epithelial phenotype and suppressing pluripotency factors to maintain a differentiated state via miR-145 and p21 [29, 64]. However, loss or inactivation of p53 suppression on pluripotent genes would result in activation of EMT and stemness factors. GOF mut-p53 further promotes EMT and stemness phenotypes by activating genes regulating them. For example, mut-p53 (R175H, R248W, R273H) was found to suppress miR-130b expression by binding to its promoter, thereby upregulating Zeb1 expression and promoting stemness via Zeb1-mediated Bmis expression (Figure 2) [48, 65]. miR-194 is another p53-responsive miRNA and represses the expression of Bmi1 oncogene that mediates pluripotency. Mut-p53 suppresses miR-194 and leads endometrial cancer cells to EMT and cancer stemness [64]. It has been found that mut-p53 (R273H) upregulates the expression of lncRNAs (lnc273–31, lnc273–34) and is implicated in EMT and CSC maintenance in colorectal cancer cells [48]. These studies highlight that mut-p53-mediated EMT phenotype confers stemness in different cancer cell lines, however, it is still required to further explore the underlying mechanisms by which mut-p53 regulates EMT genes driving cancer stemness.

3.4 Mut-p53 enhances drug resistance of cancer stem cells

A major GOF endowed by mut-p53 to cancer cells is drug resistance. Mut-p53 restrictively modulates various cellular pathways and advances cell resistance to anticancer drugs. It is more attractive to find out the specific pathways by which mut-p53 enhances the drug resistance of CSCs. For example, CSCs highly express ATP-binding cassette (ABC) transporters, that efflux drugs out of cells and confer cells resistance to chemotherapy [66, 67]. Interestingly, MDR1 (multidrug resistance 1, also named P-glycoprotein) that is the most common protein detected in tumors with drug resistance remains suppressed by wt-p53 in normal cells, but is stimulated by mut-p53 in cancer cells during tumorigenesis and associated with poor progression [68, 69, 70]. wt-p53 suppressed the expression of ABCG2 (also named breast cancer resistance protein, BCRP), which highly expresses and protects adult stem cells from drugs or toxins, but through NF-κ B pathway, ABCG2 is expressed in breast cancer cells [67, 71]. Under induced DNA damage, p53 is stabilized by feedback regulation in normal cells and it triggers cell death by apoptosis. However, this regulation mainly via p53/ubiquitin-mediated degradation is completely lost in mut-p53 cancer cells. Under DNA damage conditions, GOF mut-p53 increases ephrin-B2 expression, which in turn induces ABCG2 expression [72]. In addition, GOF mut-p53 augments the expression of anti-apoptotic proteins (Bcl-2, Bcl-xL) and represses pro-apoptotic proteins (Bax, Bad, Bid) [73]. Similarly, CSCs suppress the levels and function of Bcl-2 family proteins (Bax, Bad) to attenuate drug-induced cell death [74]. Compared to their lack in somatic cancer cells, CSCs express high levels of DNA repair genes that help them repair DNA damage inflicted by chemotherapeutic drugs [75]. It has been found that murine mesenchymal stem cells (MSCs) with mut-p53 (R172H^/+), like CSCs, express high levels homologous recombination repair and non-homologous end joining genes and induce malignant tumors in mice [76]. Also, iPS cells expressing mut-p53 (R273H, P309S) induce the expression of CD44, Lgr5, and ALDH and generate tumors [45]. Aldehyde dehydrogenase (ALDH) serves as a main marker for colorectal CSC, and notably, it is a detoxifying enzyme via the NAD(P)⁺-dependent oxidation and mediates cancer drug resistance [45, 77]. CSC phenotype is extensively driven by epigenetic factors, including miRNAs and even glycosphingolipids [78, 79]. Our recent works showed that suppressing ceramide glycosylation by glucosylceramide synthase, which is one response to cell stress under chemotherapy and generates globotriaosylceramide (Gb3) and other glycosphingolipids, can decrease GOF of mut-p53, cancer stemness, and MDR1-mediated drug resistance [29, 80, 81].

4. p53 mutants and immune evasion

mut-p53 in cancer cells contributes to immune evasion by regulating the expression of immunomodulating molecules and influencing immune cells, particularly natural killer (NK) cells and cytotoxic CD8+ T-cells and immune response in the tumor microenvironment (TME).

4.1 Mut-p53 modulates the expression of immunomodulatory ligands to affect immune response

p53 controls the expression of numerous genes, including TRAIL, DR5, TLRs, Fas, PKR, ULBP1/2, and CCL2 as well as T-cell inhibitory ligand PD-L1, which are involved in the immunological response to cancer [82]. TRAIL is expressed by several immune cells like NK cells, NK T-cells, T-cells macrophages, and dendritic cells (DCs). TRAIL binds to DR5 to cause apoptosis in a wide range of cancer types while maintaining cancer cell specificity, making it an attractive target for combination with immunotherapy [83, 84]. Toll-like receptor (TLR) 3, 5, 7, 8, and 9 play a major role in the innate immune response and stimulate the synthesis of type I interferon (IFN) via IFN regulatory factors (IRFs) [85]. IRF5 and IRF9 can be activated directly by p53 and IRF5 can induce release of cytokines leading cancer cells to apoptosis [86]. The Fas receptor defects can cause loss of immune tolerance, an accumulation of CD4⁻CD8⁻ T-cells, and production of autoantibodies [87, 88]. Under genotoxic conditions, dsRNA-activated protein kinase (PKR) regulated by p53 contributes to p53-mediated tumor suppression, also mediates inflammatory signals to activate inflammasome and proteins [89, 90]. NK cells have NKG2D receptors, which bind to ULBP1/2 ligands on the surface of tumor cells. Direct p53-target genes are ULBP1/2 ligands that improve NK cell-mediated target cell identification [14, 91]. Chemokine ligand 2 (CCL2, MCP-1) is a potent chemokine for monocytes and other immune cells. Much evidence indicates that CCL2 in the tumor microenvironment (TME) plays an immunosuppressive role [92]. GOF mut-p53 can upregulate the expression of CCL2 and tumor necrosis factor α (TNF-α) via nuclear factor kappa B (NFκB) signaling, consequently increasing microglia and monocyte-derived immune cell infiltration [93]. Several studies exhibit a correlation between mut-p53 and lack or decrease of cytotoxic immune cells.

4.2 Mut-p53 defects immunosurveillance via inactivation of NK cells

p53 maintains the immunosurveillance that recognizes neoplastic cells and initiates immune elimination; however, mut-p53 allows immune evasion and cancer progression [94, 95]. Immunosurveillance, either innate or adaptive immune responses, is composed of and relies on CD4⁺ T helper (Th) cells, CD8⁺ cells, natural killer (NK) cells and, in some cases, neutrophils [96, 97]. Among immune cells, NK cells distinguish tumor cells from normal cells mainly by relying on a balance of inhibitory and activating receptors in these cells. In brief, inhibitory receptors, such as the killer immunoglobulin-like receptors (KIR) of NK cells, recognize the major histocompatibility complex class I (MHC-I) molecules that are highly expressed in normal cells and prevent them from immune attack. Conversely, tumor cells often down-regulated express MHC-I molecules and further induce the engagement of activating receptors of NK cells, including natural cytotoxicity receptors (NCR) and the NK group membrane D (NKG2D) [98]. The cytotoxicity of NK cells is regulated by signals on both the NK cells and the targeted tumor cells. wt-p53 can upregulate the expression of ULBP1 and ULBP2 ligands on cancer cells to activate NK cells via activation of NKG2D receptor and enhance the antitumor functions of NK cells (Figure 3) [14, 99, 100]. Murine models showed that the mut-p53 (G242A corresponding to the G245A in human) suppressed the expression of active NKG2D ligand Mult-1 (while increasing the inhibitory ligand H60a) and reduced host NK cell population and activation, allowing breast tumor evade immune attack (Figure 3) [14]. Reactivating p53 function with CP31398 in breast cancer cell lines carrying mut-p53 (R280K, L194F, R175H) activated NK cells and killed cancer cells by granzyme B or NK cell-induced apoptosis [101].

Figure 3.

p53 modulates immune response combating tumor. p53 maintains immune surveillance and immune attack against cancer cells. In addition to the effects of MHC-I molecules, tumor cells expressing wt-p53 can present the ULBP1/2 surface ligands to activate host NK cells via NKG2D receptors. These wt-p53 tumor cells suppress PD-L1 inhibitory ligands, thus MHC-I molecules activate CD8+ T-cells. Activated NK cells and CD8+ T-cells excuse anticancer immunity by cytokines and induced cell death (IFN-γ, perforin, granzymes). Tumor cells expressing Mut-p53 deactivate NK cells by soluble NKG2D ligands (sNKG2D-ls) and deactivate CD8+ T-cells by overexpression of PD-L1 ligand, thus resulting in immune evasion and tumor progression. Neoantigen expressed by Mut-p53 tumor cells is weak immunogenic for activating CD8+ T-cells.

4.3 Mut-p53 proteins serve as neoantigens mediating CD8 T-cells

p53 is a tumor antigen that can differentiate cancer cells from normal cells. Recently, numerous studies showed that missense mutations of p53 in cancer cells generate neoantigens that can improve response to immunotherapy [102, 103, 104]. Tumors that express immunogenic mut-p53 (Y220C, G245S) peptides have higher expression of programmed cell death ligand 1 (PD-L1) and higher levels of tumor-infiltrating cytotoxic T-cells, as compared to tumors with wt-p53 [104, 105]. The relative contribution of mut-p53 neoantigen and immune suppression to the overall state of the TME varies across cancer types. Identifying personalized clinical approaches to targeting mutant p53 to stimulate the immune response requires careful investigation.

Recognition of MHC-I peptides by T-cells receptor (TCR) can primarily activate T-cells and the activated effector T-cells including CD4 or CD8 further upregulate co-inhibitory receptors, such as PD-1 (also known as PDCD1), to keep protective immunity in check [106]. Cancer cells can overexpress co-inhibitory ligands (such as PD-L1) to constrain T-cell activity [107]. PD-L1, an immune checkpoint protein expressed by cancer cells or other host cells binds to the programmed cell death protein 1 (PD-1) on cytotoxic CD8⁺ T-cells and results in T-cell inactivation [108]. p53 repressed the expression of PD-L1 via miR-34a in non-small cell lung cancer, and loss of p53 activity increases PD-L1 surface expression, which can suppress T-cell function and result in immune evasion (Figure 3) [109]. Further, mut-p53 (R172H) correlates with increased PD-L1 expression in lung cancers and that may help to identify patients responsive to checkpoint inhibitors targeting PD-L1 (Figure 3) [105, 109, 110, 111].

4.4 Activation of p53 reverses immunosuppression within tumor microenvironment (TME)

Regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs), and type 2 macrophages (M2) within the TME sustain pro-tumor inflammation and immunosuppression [112]. Tregs permit tumor growth by suppressing the activity of CD4+ T, CD8+ T, macrophages, and other polymorphonuclear cells (PMNs) [113, 114]. Tregs can promote angiogenesis, metastasis, and immune suppression through modulation of suppressive cytokines and surface ligands [115, 116]. p53 deficiency in prostate, ovarian, and subcutaneous pancreatic cancer can increase Treg cell populations, which are involved in suppressing effector T-cells in tumors and in the periphery [114, 117]. Mutant p53 encourages tumorigenic TGF-β signaling, which influences B cells, T regs, CD8+ and CD4+ T-cells in a variety of ways to stimulate immunosuppression [118, 119]. Activation of p53 can decrease Treg population and enhance T-cell-mediated tumor cell killing [117] and/or reverse an immunosuppressed TME by eliminating MDSCs through triggering cell death and/or reversing their immunosuppressive ability [120]. p53 can also be regulated by cytokine signaling, consistent with the observation that persistent inflammation causes stress that contributes to both tumorigenesis and tumor progression.

5. Restoration of p53 to suppress tumor progression

Mut-p53 promotes tumorigenesis and cancer progression not only because loss of wt-p53 but also the dominant-negative activities of mut-p53, which execute oncogenic GOF [22, 31, 121]. Since mut-p53 occurs in ~50% of human cancers with poor prognosis, barely in normal tissues, it has been emerged a specific therapeutic target. Approaches for directly targeting mut-p53 mainly include: (1) restoration of the DNA-binding conformation; (2) depletion or degradation of mut-p53 proteins; and (3) epigenetic restoring wt-p53 expression. More information regarding clinically available FDA-approved drugs and drugs in clinical trials for targeting mut-p53 or restoring p53 functions can be found in recent reviews [22, 122].

5.1 Reactivation of the transcriptional activity

mut-p53 proteins alter the expression of target genes transcriptionally and the conformation for protein-DNA has been recognized as crucial step [5]. Reactivating the transcriptional activity of mut-p53 is an effective strategy to eradicate cancer in 20 years. Many studies found that small molecule compounds and peptides can induce changes in the spatial conformation and folding pattern of mut-p53, are referred to reactivators (Table 2, Figure 4).

Regimen	Target spectrum	Indications	Clinical Trials
Res. Transcriptional Activity
APR-246 alone or plus others (azacitidine, pembrolizumab)	R175H, R273H by the thiol groups binding	Multiple tumors	~ 13 trials in Phases I, I/II, III
ADH-6, ReACp53	R175H, R248, R273 by inhibiting aggregation	Cancer cells	n/a
PC14586	Y220C by	Solid tumors	Phase I/II
Arsenic trioxide (ATO) ATO + decitabine	R175H G245, R249, R282 by rescue functionality	Tumors, AML, MDS; MDS, AML	~5 trials; Phases I, I/II, III
C-peptide (361–382), pCAPs; N-peptides	R273H and others; Prevent MDM2 binding	Cancer cells	n/a
Degrading mut-p53
Ganetespib + paclitaxel Ganetespib + docetaxel	R175H, R248Q by HSP90 inhibition	Ovarian cancer; NSC lung cancer	Phase I/II; Phase III
Vorinostat + MLN9708; Vorinostat + pazopanib	R175H, R273H by HDAC6 inhibition	Solid tumors; Advanced cancer	Phase I; Phase I
MCB613	R175H by inhibiting deubiquitinase USP15	Ovarian cancer cells	n/a
Restoring p53 expression
C6-ceramide,; Cer-RUB nanomicelles; PDMP, Genz667161	Deletion; R273H, R238W by m6A-mediated pre-mRNA splicing	Colon cancer cells; Ovarian cancer cells	n/a
Neplanocin A	R273H by m6A inhibition	Colon cancer cells	n/a

Table 2.

Targeting p53 mutants directly to improve cancer treatments.

PDMP and Genz667161 are GCS inhibitors.

Figure 4.

Restoration of p53 in tumor suppression. Cancer cells dominantly express mutant p53 proteins from mRNA (wt, Mut) and promote tumor progression. The transcriptional activity of mutant p53 in tumor suppression can be restored directly by: (1) reactivating p53-transactivity. Small molecules modulate the conformation of Mut-p53 protein to increase transcription of p53-responsive genes. (2) degrading Mut-p53. Small molecules modulate signaling pathways to increase the ubiquitination of Mut-p53 proteins. (3) restoring wt-p53 expression. m⁶A, N⁶-methyladenosine; wt, wild-type; Mut, mutant; GOF, gain-of-function.

Restoring p53 binding: Among small molecules of reactivators, the most representative one is APR-246 (also known as eprenetapopt or PRIMA-1^MET), which is involved in more than 13 clinical trials, including phase 3 (Table 2). As a novel and more effective reactivator, prodrug APR-246 is converted to a Michael acceptor methylene quinuclidinone (MQ) that binds covalently to the cysteines, leading to refolding protein and restoring the activity in transcription of p53-target genes [123, 124]. Consistent with its capacity of reactivating mut-p53 (R175H, R273H), the anticancer effects of APR-246 have been demonstrated in a range of in vitro and in vivo preclinical cancer systems [124, 125]. In cancer patients, APR-246 monotherapy has been shown to induce p53-dependent biologic effects against malignancy. In clinical trials enrolling patients with cancers, APR-246 was found to be safe and to have therapeutic activity when combined with particular medications, like azacitidine or pembrolizumab (targeting PD-1) [22]. The FDA granted breakthrough designation to APR-246 treatment for myelodysplastic syndrome (MDS), and a phase III randomized trial for the combined treatment was initiated in multicenter (NCT03745716). Recently, a phase II trial announced encouraging results, for evaluating APR-246 plus azacitidine as a post-transplant management therapy in patients with MDS and AML with mut-p53. Combined treatment with APR-246 and azacitidine achieved significant increased relapse-free survival (RFS, 58 vs. 30%) with median overall survival (OS, 19.3 vs. 5–8 months) after transplantation, compared to previous trials [126].

Decreasing mut-p53 Aggregation: Tripyridyl amide ADH-6, which inhibits amyloid formation with Alzheimer’s disease, was identified as an inhibitor of mut-p53 aggregation. ADH-6 enhances cell death and inhibits tumor growth, with high selectivity for cancer cells expressing mut-p53 (R175H, R248W/Q , R273C/H), no toxicity to healthy tissues, p53-null cells or cells with wt-p53. ADH-6 has a greater efficacy than ReACp53, which is a cell-penetrating peptide for inhibiting mut-p53 (R282W) amyloid formation [127, 128]. In this group, none of the agents have been applied in clinical trial. Whereas, above compounds are relatively broad spectrum, further studies aimed at targeting specific single mutant or a distinct group of similar mutants effectively should be pursued.

Specific Codon Revealing: The small molecule PhiKan083 as well as PK7088 was found to bind the crevice near the mutant site and thermodynamically stabilize mut-p53 (Y220C), shifting it toward a wt-p53-like state [129, 130]. Compound PK7088, which is cooperated with nutlin-3 (wt-p53 activator) to transactivate p53-target genes, induced apoptosis and decreased the viability of gastric cancer and hepatoblastoma cells that express mut-p53 (Y220C) [130]. The p53 (Y220C)-specific small molecule, PC14586, which is bioavailable orally and is now in phase I/II clinical trial (NCT04585750), has presented highly promising results [131, 132]. The discovery of p53 (Y220C)-specific drugs is encouraging, however, the p53 Y220C is not a very common mutant (~0.64% of cancer cases) and has a unique structure not shared with other mut-p53 proteins.

Stabilizing Conformation of DNA binding: The mut-p53 (R175H) results in impaired zinc binding, causing misfolding and inactivation of the p53 protein. NSC319726 (also known as ZMC1), a metal ion chelator with high affinity for zinc, has been identified as a mut-p53 (R175H)-targeting drug [133]. NSC319726 promoted p53-dependent apoptosis and tumor regression in vivo, in a manner highly specific to cancer cells carrying p53 R175H [133, 134]. It has been found that ZMC2 and ZMC3, which belong to the same family of thiosemicarbazones to rescue conformation via zinc, promote a wt-like conformation into mut-p53 (R175H) in vitro [134]. COTI-2, a third-generation thiosemicarbazone now in clinical trial phase I, showed preferential selectivity for cancer cell lines expressing p53-mut (R175H), but also had some activity in wt-p53 cells [135]. Interestingly, it was proposed that COTI-2 triggers cell death by p63 (a p53 family member), which activates p53-target genes under DNA damage and replication stress [136]. Arsenic binding can stabilize the DNA-binding loop-sheet-helix motif alongside the overall β-sandwich fold, endowing mut-p53 with transcriptional activity [137]. Arsenic trioxide (ATO; Trisenox) is an FDA-approved drug for the treatment of acute promyelocytic leukemia. Through in silico analysis, it was found that ATO can rescue the functionality of mut-p53 with only a limited effect on DNA contact mutants [137]. ATO can restore the proper folding of several mut-p53 (R175, G245, R249, R282), however, only a subset of those regain wt-p53 transcriptional activity [137]. ATO is involved in several trials, treating patients with TP53-mutated AML, multiple tumors, and MDS alone or with decitabine (Table 2).

Peptides Competitive Binding: Both N- and C-terminal peptides have been produced to improve or restore p53 function in mut-p53 status. To disrupt the C-terminal negative transcriptional regulatory element of p53, a series of carboxy-terminal peptides were employed [138, 139, 140]. Among them, in some mut-p53 containing cell lines, a peptide (from p53 protein-seq 361–382) showed a strong potential for restoring DNA binding and transcriptional activity to altered p53. The effect was dependent on mutant p53 expression, and the peptide had no toxic effect on wt-p53 or p53-null cells. Further study revealed that the peptide not only binds to the C-terminus but also to the central domain, thus inducing Fas/APO-1-mediated apoptosis [140]. A peptide that binds to and stabilizes the p53 core domain can function as a chaperone, restoring a wt-like conformation [141].

p53 N-terminal peptides have also been generated to prevent MDM2 binding to p53, and so liberate p53 from MDM2-mediated proteasomal degradation. Two peptides that resemble the N-terminus of p53 can attach to MDM2 with an affinity that is 100 times greater than p53, thus preventing MDM2-mediated p53 degradation. Cells that have wt-p53 experience apoptosis after being treated with the peptides [138].

A panel of mut-p53 reactivating small peptides (pCAPs) that were developed using phage display selection showed p53-dependent effects in vitro and in vivo when applied to cancer cells expressing mut-53. Mechanistic studies suggested that pCAPs bind preferentially to the wt-p53 conformation; when a mut-p53 molecule assumes transiently a wild-type-like conformation, the peptide binds and stabilizes it, gradually shifting the dynamic equilibrium of the p53 population in that direction [142].

5.2 Degradation of Mut-p53

TP53-mutated cancer cells are addicted to mut-p53 proteins that execute oncogenic GOF [143]. Decrease of mut-p53 proteins in cancer cells by promoting degradation exhibits antitumor effects (Figure 4) [144, 145]. Some drugs such as gambogic acid [146], capsaicin [147], MCB-613 [144], and NSC59984 [148] can induce the degradation of mut-p53 in several different types of cancer cells. Among these agents, compound MCB-613 that inhibits deubiquitinase USP15 causes rapid ubiquitination, nuclear export, and degradation of mut-p53 (R175, less effects to others) in ovarian cancer cells [144].

Inhibition of HDAC6/HSP90 chaperone axis. Recent studies found that the accumulation of mmt-p53 (R175H, R273H) depends on the histone deacetylase 6 (HDAC6)/HSP90 chaperone axis that is sustained by RhoA geranylgeranylation downstream of the mevalonate pathways [145, 149]. Ganetespib (formerly known as STA-90909), a potent HSP90 inhibitor synergizes with cyclophosphamide to improve survival of mice with autochthonous tumors in a mutant p53-dependent manner [150]. Treatment of mut-p53 mice (R172H, R248Q^/−) with ganetespib inhibits tumor growth and prolongs survival in a mut-p53 dependent manner. Currently, the suberoylanilide hydroxamic acid (SAHA, a HDAC inhibitor) and ganetespib are in clinical trials (Table 2). Vorinostat (a HDAC inhibitor) with pazopanib is used as a therapeutic approach for inhibiting p53-mediated angiogenesis and facilitating mutant p53 (R175H, R273H) degradation in a phase I study. This evidence supports further evaluation of the combined treatment in patients with mut-p53 cancers, especially metastatic sarcoma or metastatic colorectal cancer [151].

5.3 Restoration wt-p53 protein expression

TP53 mutation is usually heterozygous in either germline or somatic cells, including cancer cells [8, 25, 30]. This may provide opportunities to modulate the transcriptional and post-transcriptional processes to restore wt-p53 expression in mut-p53 cancer cells. Restoration of wt-p53 expression with depletion of mut-p53, which avoids the adverse effects of heterotetramer in cancer cells, would be more effective than other strategies for targeting mut-p53 (Figure 4) [121]. It is an intriguing finding that suppression of glucosylceramide synthase (GCS) restores p53-dependent apoptosis in ovarian cancer cells expressing deletion-mutant p53 (6 amino acids in exon-5) [30]. Inhibition of GCS eliminates the oncogenic function of mut-p53 (R273H) in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells [29]. GCS converts ceramide into glucosylceramide and regulates the synthesis of glycosphingolipids, including Gb3 [152]. Suppression of GCS, by either silencing its expression or inhibiting its activity with PDMP and Genz667161, restores p53-dependent tumor suppression [29, 30, 80, 153]. It is ceramide that can restore the expression of wt-p53 via SRSF1-mediated RNA splicing in these deletion-mutant cancer cells [154]. Our further works indicate that N⁶-methyladenosine (m⁶A) catalyzed by methyltransferase like-3 (METTL3) at the mutant codon promotes the expression of mut-p53 (R273H) and its GOF by enhanced m⁶A-mRNA splicing [155]. Decreased METTL3 expression by treatments with small interfering RNA (siRNA) or with neplanocin A (NPC) restored wt-p53 expression and function in suppressing tumor growth (Table 2) [155]. These clearly indicate that m⁶A at the mutant codon of pre-mRNA determines the protein expression of mut-p53 (R273H). Interestingly, either the suppression of ceramide glycosylation with GCS inhibitors (PDMP, Genz667161) or the provision of cell-permeable ceramide (C6-ceramide, Cer-RUB nanomicelles) can restore p53-dependent functions in eliminating CSCs, reverse of drug resistance, and decrease of tumor growth of ovarian cancer and colon cancer cells carrying mut-p53 (R273H, R248Q) (Table 2) [29, 58, 80]. These are proof of concept for restoring wt-p53 expression and its tumor suppression to target cancers expressing mut-p53.

6. Conclusion

Recent progression in p53 and cancer studies has characterized the critical role played by GOF mut-p53 in cancer stemness and immune evasion. GOF mut-p53 can reprogram and dedifferentiate cancer cells or normal epithelial progenitors by overexpression of pluripotent factors and forward activate the downstream signaling pathways, thus promoting cancer stemness. Cancer cells expressing mut-p53 can remarkably deactivate NK cells or CD8+ T-cells so as sheltering tumors against immunosurveillance and immune attack. These situations could be clearly observed in tumors or cancer cells under cell stress, such as chemotherapy or radiation therapy, within mut-p53 is overexpressed. Altogether, targeting mut-p53 would be more effective means than others to eliminate cancer progression and recurrence. Discovery of small molecule drug restoring p53 function is showing increasing promise, even major challenges still remain and multiple clinical trials are attempted to bring such molecules into the clinic. It is realized that the cellular processes and underlying regulatory mechanisms for the expression and degradation of mut-p53 proteins are distinguished from those for wt-p53. Enhancing ubiquitination to degrade mut-p53 and modulating m6A methylation-RNA splicing to restore wt-p53 expression are emerged as feasible approaches for targeting cancers expressing mut-p53.

Specifically, targeting mut-p53 proteins and the oncogenic GOF is critically important for improving the outcomes of cancer treatments. Further understanding in how mut-p53 executes oncogenic GOF in cancer stemness and how mut-p53 regulates particular ligands of CSCs to modulate host immunosurveillance and immune response, would help us to discover combined approaches against CSCs of tumors expressing mut-p53. Understanding how ubiquitination and underlying signaling pathways to degrade mut-p53 proteins, rather than wt-p53 protein in cancer cells, would help to develop new compounds or FDA-approved drugs to more effectively restore p53-dependent tumor suppression. More importantly, understanding m6A methylation at mutant codon of p53 pre-mRNA and further alternative RNA splicing process and the underlying mechanisms would help us to selectively target mut-p53 and more effectively combat most cancers.

Conflicts of interest

YY Liu is a member of Scientific Advisory Board for Sanofi-Genzyme and Board of Director of Mycobacterium DX Research Lab, and no other authors have competing interests.

Funding

The present study was supported by an Institutional Development Award (IDeA) from the National Institute of General Medicine Sciences of the National Institutes of Health under grant number P20 GM103424–21 (to Y.Y.L.).