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    Topic review

    PI3K/Akt/PTEN Dysregulation in Canine Tumor

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    Submitted by: Jung Hyang Sur

    Definition

    PIK3CA H1047R mutation was detected in 14.3% (10/70) of tumor samples. Dysregulation of p-Akt, Akt2, and PTEN was observed in mammary tumor samples, but only PTEN dysregulation was associated with PIK3CA H1047R mutation. Conclusions: The present study showed that dysregulation of components in the PI3K/Akt/PTEN pathway is a feature of canine mammary tumors, but this dysregulation is not directly correlated to the PIK3CA H1047R mutation except for PTEN expression

    1. Introduction

    PI3K (phosphoinositide-3-kinase) was first discovered as a viral oncoprotein that phosphorylates phosphatidylinositol, induces the transformation of cells, and has been revealed to be conserved in mammals [1].
    PIK3CA is one of the catalytic subunits of the class I PI3K subfamily and encodes a catalytic subunit p110α that functions as a heterodimer with the p85 regulatory subunit [2]. Class I PI3K is stimulated by numerous signals received from tyrosine kinase receptors, cytokines, and G protein-coupled receptors [3][4]. In response to the signals, PI3K phosphorylates lipids in plasma membrane, phosphatidylinositol-4,5-biphosphate to phosphatidylinositol-3,4,5-trisphosphate [3][4]. The lipids that are produced in this reaction interacts with the v-Akt murine thymoma viral oncogene homolog (Akt) pleckstrin homology domain, and consequently, phosphorylated Akt plays a key second messenger to various cell signaling [3][4]. In addition, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) dephosphorylates the lipid phosphatidylinositol-3,4,5-trisphosphate, which is the product of PI3K, thus hindering the activation of Akt and acting as a tumor suppressor [5].
    Members of the PI3K family play active roles in a wide range of physiologic processes, thus making the dysregulation reasonable in several diseases including diabetes, neurological, and immunological disorders [6]. The role of PI3K has been highlighted especially in oncogenesis, because overactivation of PI3K with enhanced Akt activity and PTEN suppression is associated with most hallmarks of cancer [6][7]. For instance, overactivation of the PI3K/Akt pathway can induce the progression of cell cycle and cellular proliferation through stability regulation of cyclin D and p21Cip1 and can inhibit apoptosis by modulating the activity of Bcl-2 family members [8]. Moreover, PI3K signaling contributes to cell migration and migratory cell polarization in various cell types [9]. Overall, the frequent activation of the PI3K pathway with diverse contribution to oncogenesis makes it an attractive therapeutic target [10].
    Specific mutations in PI3KCA have been identified in various tumors from 2004 [1][11][12], and intense studies investigating the role and regulation of PIK3CA have been progressed. Mutations in the PIK3CA gene were found in a wide range of human cancers including glioblastoma [12][13], gastric cancer [12][14], lung cancer [12][15], colorectal cancer [16], and breast cancer [11][12]. In human breast cancer, somatic mutation of PIK3CA has been found in 8–40% of case samples. Mutational hotspots were identified on exon 9 and exon 20, and the most frequent mutation has been exon 20 H1047R in human breast cancer, implicating that it is an oncogenic driver [11][12][17][18][19][20].
    Several investigations have proven how mutant PIK3CA H1047R specifically influence oncogenic and physiological processes in in vitro and in vivo models. The PIK3CA H1047R mutation has been known to gain-of-function mutation stimulating catalytic activity [21], and expression of PIK3CA H1047R induced tumor initiation [22], cell dedifferentiation [23], tumor heterogeneity [24], and invasiveness and migration in mammary tumor cells [25].
    In recent years, research applying next-generation sequencing has opened a new landscape in veterinary oncology. PIK3CA has started to be highlighted because its frequent mutations in canine tumors have been revealed. The latest studies have shown that single missense mutation H1047R is the feature that is most discovered in canine hemangiosarcoma [26] and mammary tumors [26][27][28].
    There are few studies exploring the expression patterns of the PI3K pathway in canine mammary tumors, reporting the upregulation of phospho-Akt (p-Akt) and loss of PTEN is related to histologic and clinical malignancy [29][30]. However, research examining the correlation between H1047R mutation and the expression of PI3K/Akt pathway molecules is missing in the literature. Therefore, the current study aims to analyze the frequency of the PIK3CA H1047R mutation in canine mammary tumors and to investigate the correlation between PIK3CA H1047R mutation and the factors including the PI3K/Akt/PTEN pathway components’ expression, histopathological features, and clinical characteristics.

    2. Discussion

    In this study, we found that components of the PI3K/Akt/PTEN pathway—p-Akt, Akt2 and PTEN—were dysregulated and differentially expressed depending on histological grade. The current study also revealed that the PIK3CA H1047R mutation is a relatively frequent event in canine mammary tumors, and this mutation was mostly not correlated with downstream molecule expressions or histopathological/clinical features. In human breast cancers, PIK3CA mutation is associated with PTEN loss and Akt activation represented by p-Akt [31][32]. These dissimilarities may suggest that different mammary tumorigenic mechanism in humans and dogs, for example, activation of Akt may be PIK3CA-independent in canine mammary tumors, possibly phosphorylated by tyrosine or serine/threonine kinases [33]. However, the greater possibility for this discrepancy could be derived from insufficient numbers of PIK3CA-mutated samples (n = 10) in this study to correctly determine the feature of PIK3CA-mutated tumors. Because studies verifying canine PIK3CA H1047R are still limited and the numbers of cases in previous and the present study were relatively low, further large-scale studies are required to determine the frequency and the features of PIK3CA H1047R mutation in canine mammary tumors.
    While PIK3CA H1047R missense mutation is a frequent event in canine mammary tumors in recent studies utilizing next-generation sequencing (29–32.8%) [27][28], mutation was observed in relatively low occurrence in the present study (14.3%). Low percentage of PIK3CA H1047R could be derived from the difference of sensitivity between next-generation sequencing and Sanger sequencing. As conventional Sanger sequencing needs 20% of allele frequency to detect the mutation and as mammary tumor is highly heterogenous [34], samples harboring low mutant allele could be false-negative in the present study.
    Downregulation of PTEN in malignant tumors in this study corresponded to previous findings, emphasizing its role in canine mammary tumorigenesis [29][30][35]. PTEN loss is also a common feature in human cancer, derived from epigenetic silencing, mutation, and transcriptional repression [36]. Since PTEN is a multifaceted molecule associated with cell cycle, cell motility, genomic stability, and tumor microenvironment [36], and consistent results are shown in previous and current study, investigating PTEN in combination with other molecules could be beneficial to enhance our understanding of canine mammary tumors. Lower expression of PTEN in simple-solid carcinoma than in complex adenoma and complex carcinoma is also an interesting finding as has been shown in a previous study [29]. From the result of the previous and current study, it might be speculated that the reason why dogs with complex-type tumor survive longer than dogs with simple-type tumor [37] originates from the dysregulation of PTEN. From this point of view, PTEN itself could be a potential prognostic marker in canine mammary tumors. In addition, paradoxical higher expression of PTEN in PIK3CA-mutated tumors in the current study may be a true feature of PIK3CA-mutated tumors, however, it may be derived from the small sample size of PIK3CA-mutated tumors (n = 10) to correctly deduce the conclusion.
    In the present study, the entire expression of Akt was similar in normal and tumor tissues, whereas contrasts in p-Akt and Akt2 expression were demonstrated in different histological grades. Thus, it could be concluded that although transcriptional and translational levels of Akt are not changed in tumors, activation of Akt and expression of Akt isoforms are dysregulated. Despite there being a conflicting result [29], activation of Akt is a seemingly early event in the transformation in canine mammary tumors as upregulation is shown from adenoma.
    Moreover, as Akt2 has been demonstrated to promote tumor invasion and cell migration [38], significant upregulation of Akt2 transcription found in the present study may imply that overexpression of Akt2 in high-grade tumors constitutively function as a key factor for aggressiveness in canine mammary tumors. The previous [27][28] and the present study suggest that PIK3CA H1047R frequently occurs in canine mammary tumors. This mutational similarity to humans may shed light on new anticancer therapy in dogs. PI3K signal promotes the growth of estrogen receptor-positive breast cancer in an estrogen-independent manner, and blocking of PI3K inhibits the emergence of hormone-independent cancer cells [39]. PIK3CA mutation has been found to be common in canine mammary tumors and carcinogenesis of canine mammary tumor is estrogen-dependent with estrogen receptor expression [40]. Thus, PIK3CA could be suggested as a potential therapeutic target. For example, Alpelisib, which is a new anticancer drug and is clinically beneficial to PIK3CA-mutated breast cancer patients [39][41], might be a future medicine for dogs with mammary tumors.

    3. Conclusions

    Overall, the present study examined the dysregulation of PI3K/Akt/PTEN axis molecules and the PIK3CA H1047R mutation by utilizing Sanger sequencing in canine mammary tumors. PIK3CA, which has started to be highlighted in recent years, has undeniable worth in understanding molecular pathogenesis for canine tumors, and could be a potential therapeutic target in canine mammary tumors. Therefore, further large-scale studies investigating thee PIK3CA/Akt/PTEN pathway in canine mammary tumors are necessary.

    The entry is from 10.3390/ani11072079

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