The management of NMIBC persists as a challenge due to the high rates of recurrence and progression to muscle invasive stage. BCG use in NMIBC is limited by treatment failure, adverse effects and intolerance that appear on two-thirds of patients [4, 22, 23] (Herr et a., 2015; Lamm et al., 2000; Ojea et al., 2007). Metabolic disorders, such as diabetes mellitus type 2, could contribute to the failure of BCG in NMIBC [24](Ferro et al., 2020). In cases of BCG treatment failure, radical cystectomy is the only available therapeutic option. However, in patients ineligible for radical cystectomy, the treatment possibilities are limited [4, 22, 25] (Herr et al., 2015; Lamm et al., 2000, Witjes, 2006).
In view of these problems and limitations associated with the use of BCG, novel therapies are needed to treat NMIBC and prevent disease progression while preserving bladder function and ensuring acceptable quality of life for patients. These new therapies would also supply an alternative for those ineligible for radical cystectomy. P-MAPA, a biological response modifier that shows novel therapeutic properties compared to standard treatments, appears to be a valuable candidate drug for the treatment of NMIBC. In previous reports, we described several therapeutic properties of P-MAPA.[9, 10, 12], (Fávaro et al., 2012; Garcia et al., 2015; Garcia et al., 2016) Although all the studies performed in animal models to assess the therapeutic potential of P-MAPA on NMIBC compared with BCG does not involve tumoral resection (TURBT) due experimental restrictions, all of them, used a model of tumoral induction based on intravesical instillations of MNU. As reported in the literature [26] (Weldon and Soloway, 1975) the use of MNU like TURBT, lead to an altered urothelial surface, favoring the implantation of tumoral cells, or in other words, the use of intravesical MNU may result in an altered presentation of urotelial surface similar to those induced by TURBT.
Here, by using the NMU rat model to study NMIBC, we found that treatment with BCG led to histopathological recovery in only 20% of the rats. In contrast, intravesical immunotherapy with P-MAPA provided better histopathological recovery from cancer in relation to BCG treatment (MNU-BCG group).
Few years ago, an important research and strategies for the cancer therapy ( e.g., NMIBC), mainly analyzing molecules that bind to and activate TLRs were studied [10, 12, 27] (Garcia et al., 2015; Garcia et al., 2016; LaRue et al., 2013). Our previous knowledge with animal model on NMIBC, P-MAPA increased TRIF and IRF-3 protein levels, indicating an activation of MyD88-independent pathway [9, 12] (Fávaro et al., 2012; Garcia et al., 2016). P-MAPA induction of MyD88-independent pathway, which is a TRIF-dependent pathway, produced an enhancement of IFN-γ and iNOS (macrophages type 1 – M1) protein contents. P-MAPA immunotherapy led to distinct activation of innate immune system of TLRs 2 and 4-mediated, differently to BCG, overcoming in an augmented interferons signaling pathway [12] (Garcia et al., 2016), giving a more efficient effect in the NMIBC treatment.
IFN-γ and IRF-3 induction (interferon signaling pathwas) by P-MAPA, when compared proliferation/apoptotic ratio in the animal treatment by P-MAPA to BCG was significantly lower in the animals treated with the former than with BCG treatment, denoting prevalence of the apoptotic pathway [12] ( Garcia et al., 2016).
Thereby, P-MAPA on activation of interferon signaling pathway was highly more effective in the enhancement of immunogenic cell death in relation to inflammatory cytokines signaling pathway through BCG. Thus, P-MAPA is considered as a cytotoxic immunotherapy [9. 12] (Fávaro et al., 2012; Garcia et al., 2016).
P-MAPA immunotherapy also increased p53 levels [9, 12]. (Fávaro et al., 2012; Garcia et al., 2016). The expression of proteins and inflammatory cytokines is altered after p53 activation in cells related with immune system [28] (Shatz et al., 2021). TLRs expression was altered in cancer cell lines after p53 activation, resulting in increased ligand-mediated expression of cytokines downstream from the corresponding TLR [29, 28] (Menendez et al. 2013; Shatz et al., 2012).
The modulation of TLR expression in tumors associated with immune cells in response to DNA-impairing and/or p53-inducing agents could be useful in approaches that use TLRs for cancer management [13, 20] [Menendez et al., 2011; Li et al., 2011). Studies demonstrated that wild-type and p53 mutants modulate TLR expression differentially. Shatz et al. [28] (2012) proposed that management of normal or mutant p53 responses along with immune challenges that include TLR targets could increase inflammatory and immune type responses. New treatments based on restore wild-type p53 to cancer cells, may stop or significantly reduce cancer growth [29, 30] (Menendez et al. 2013; Li et al., 2011). These treatments involve medicines that could alter “mutated p53” and make it behave as wild-type p53 [29] (Menendez et al. 2013). Furthermore, other therapies could block proteins that degrade p53 in cancer cells, resulting in increased cancer cell p53 levels with consequent cancer cell death. We have previously shown that immunomodulation of TLRs 2 and 4 by P-MAPA led to wild-type p53 activation in NMIBC [9, 12] ((Fávaro et al., 2012; Garcia et al., 2016). In addition, in our study, we have shown that intravesical immunotherapy with P-MAPA remarkably increased wild-type p53 protein levels in a rat model of NMIBC. This effect was not seen with BCG therapy, which suggested that immunotherapy with P-MAPA could be a relevant pharmacological approach for increasing wild-type p53 protein levels, in agreement with Menendez et al. [29] (2013).
In mouse fibroblasts studies have shown that induction of p53 in response to activated oncogenes such as E1A, Raf, Ras, β-catenin, v-abl, E2F1 and c-Myc involves the p19ARF protein that binds and also inhibits Mdm2 [19, 31] (Lindstrom et al., 2003; Sherr, 1998). At normal conditions, p53 is a short-lived protein because of its fast proteasomal degradation [33. 33, 34, 35] ](Wang et al.,2002; Wang et al., 2003; Vousden and Lane, 2007; Lim et al., 2009). This kind of degradation is largely mediated by Mdm2 which is one of p53's own target gene products, that fits as an E3 ubiquitin ligase for p53, although Mdm2-independent pathways for p53 degradation also exist [32, 33. 36, 37, 38] (Wang et al.,2002; Wang et al., 2003; Ashcroft et al., 2000; Brune et al., 2001; Umansky and Schirrmacher, 2001).
The inhibition or suppression of apoptosis is considered an important factor in leading tumorigenesis in vivo [18] (Phesse et al., 2014). Although c-Myc protein can drive apoptosis in various biological levels [18, 39] (Phesse et al., 2014; Hoffman and Liebermann, 2018), the simple overexpression of c-Myc in pancreatic islets does not lead to carcinogenesis except if apoptosis is blocked, e.g., by p53 loss, p19ARF knockout or Bcl-xl overexpression [18, 40] (Phesse et al., 2014; Finch et al., 2006). Seoane and Massague [41] (2002) demonstrated that depletion of c-Myc in colorectal cancer cell lines reduced the downstream effectors of p53 signaling, resulting in a decrease in apoptosis. These authors concluded that, in the absence of c-Myc, the levels of the anti-apoptotic cell cycle arrest protein p21 were enhanced, while those of pro-apoptotic genes such as BAX were reduced, coming out in cell cycle arrest rather than apoptosis. Phesse et al. [18] (2014) elucidated the fundamental function of c-Myc in signaling DNA damage-induced apoptosis in vivo through the control of p53 protein. These authors investigated the apoptotic response to DNA damage after deleting the c-Myc gene in intestinal enterocytes from adult murine intestine and found that c-Myc deletion completely abolished the immediate wave of apoptosis that followed ionizing irradiation and treatment with cisplatin, a situation that mimicked the phenotype of p53 deficiency in the intestine. Since c-Myc-deficient intestinal enterocytes do not upregulate p53, Phesse et al. [18] (2014) concluded that these results reflected an upregulation of the E3 ubiquitin ligase Mdm2 that targets p53 for degradation in these cells. In addition, low level overexpression of c-Myc elicited persistent apoptosis in response to DNA damage, indicating that c-Myc acts as a decisive cell survival rheostat after DNA damage.
High E3 ubiquitin ligase Siah-2 (seven in absentia homolog 2) and androgen receptor protein levels play an important role in urothelial carcinogenesis, presumably leading to the escape of urothelial cancer cells from attack by the immune system [10] (Garcia et al., 2015). The latter authors also demonstrated that intravesical treatment with P-MAPA downregulated Siah-2 protein levels, an essential step for histopathological recovery from cancer. In support of Phesse et al.[18] (2014) and Garcia et al. [10] (2015), the present study demonstrated that immunotherapy with P-MAPA enhanced c-Myc protein levels in a rat model of NMIBC, resulting in the downregulation of ubiquitin ligase Siah-2 and an increase in wild-type p53 levels. As a result of the increase in wild-type p53 levels, the proliferation/apoptotic ratio was remarkably lower and the BAX protein level was significantly higher in rats treated with P-MAPA, indicating a predominance of apoptosis. We have thus identified an important mechanism of action for P-MAPA, namely the mediation of c-Myc-induced apoptosis by p53; this phenomenon was not observed after treatment with BCG.
Few years ago, the role of other factors in regulating p53 activity has been investigated, particularly that of COUP-TFII [14, 16] (Huang et al., 2004; Xu et al., 2015). The importance of COUP-TFII in p53 signaling and its interaction with c-Myc in triggering apoptotic mechanisms in NMIBC is poorly understood. Huang et al. (2004) [14] found that COUP-TFII expression induced a distinguished accumulation of p53 protein when contrast with steady-state protein levels in HCT116 cells. This finding suggested that COUP-TFII was a bona fide agonist of the p53 transcriptional network. These authors also demonstrated that expression of COUP-TFII in avian and zebrafish developmental systems activated p53 and produced apoptosis in vivo, resulting in a phenotype like to that of p53 overexpression.
When patients exhibit COUP-TFII-positive colorectal tumors they have a better overall survival rate than with those with tumor with COUP-TFII-negative [42] (Shin et al., 2009). Other work also found relatively low expression of COUP-TFII in ovarian tumors in relation to healthy tissue [43] (De Souza et al., 2007). A high COUP-TFII transcript level was related to an increased survival and its expression inhibited the TGF-β-dependent epithelial-mesenchymal transition and chemoresistance in human breast cancer [44](Zhang et al., 2014). Based on the latter results, Xu et al. [16] (2015) indicated that COUP-TFII serves either as a tumor promoter and suppressor in different tumor types. Here, we have demonstrated for the first time that COUP-TFII levels were significantly lower in NMIBC rats compared to healthy rats (control group). Intravesical immunotherapy with P-MAPA significantly increased these levels, which were like to those in the control group. In the BCG group, COUP-TFII levels were significantly lower compared to the P-MAPA and control groups. All of these findings indicated that COUP-TFII levels were associated with c-Myc in the activation of wild-type p53, resulting in enhanced apoptosis in NMIBC.
In conclusion, we have identified an important mechanism of action for P-MAPA based on an enhancement in wild-type p53 levels and the mediation of c-Myc/COUP-TFII-induced apoptosis by p53. This overall pathway was fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. The action of P-MAPA in these apoptotic pathways may represent a new strategy for treatment of NMIBC.