PI3K/Akt/PTEN Dysregulation in Canine Tumor: Comparison
Please note this is a comparison between Version 2 by Amina Yu and Version 1 by Jung Hyang Sur.

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

  • dogs
  • mammary neoplasms
  • PI3K/Akt/PTEN pathway
  • PIK3CA H1047R mutation

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][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][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][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][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[1][11][12],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[12][13],13], gastric cancer [12[12][14],14], lung cancer [12[12][15],15], colorectal cancer [16], and breast cancer [11,12][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][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][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][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 [41,42][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 [43][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[27][28],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 [44][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 [29,30,45][29][30][35]. PTEN loss is also a common feature in human cancer, derived from epigenetic silencing, mutation, and transcriptional repression [46][36]. Since PTEN is a multifaceted molecule associated with cell cycle, cell motility, genomic stability, and tumor microenvironment [46][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 [47][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 [48][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][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 [49][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 [50][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 [49,51][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.


  1. Arafeh, R.; Samuels, Y. PIK3CA in Cancer: The Past 30 Years. Semin. Cancer Biol. 2019, 59, 36–49.
  2. Jean, S.; Kiger, A.A. Classes of Phosphoinositide 3-Kinases at a Glance. J. Cell Sci. 2014, 127, 923–928.
  3. Martini, M.; De Santis, M.C.; Braccini, L.; Gulluni, F.; Hirsch, E. PI3K/AKT Signaling Pathway and Cancer: An Updated Review. Ann. Med. 2014, 46, 372–383.
  4. Paez, J.; Sellers, W.R. PI3K/PTEN/AKT Pathway. A Critical Mediator of Oncogenic Signaling. Cancer Treat. Res. 2003, 115, 145–167.
  5. Jerde, T.J. Phosphatase and Tensin Homologue: Novel Regulation by Developmental Signaling. J. Signal. Transduct. 2015, 2015, 282567.
  6. Fruman, D.A.; Chiu, H.; Hopkins, B.D.; Bagrodia, S.; Cantley, L.C.; Abraham, R.T. The PI3K Pathway in Human Disease. Cell 2017, 170, 605–635.
  7. Fruman, D.A.; Rommel, C. PI3K and Cancer: Lessons, Challenges and Opportunities. Nat. Rev. Drug Discov. 2014, 13, 140–156.
  8. Chang, F.; Lee, J.T.; Navolanic, P.M.; Steelman, L.S.; Shelton, J.G.; Blalock, W.L.; Franklin, R.A.; McCubrey, J.A. Involvement of PI3K/Akt Pathway in Cell Cycle Progression, Apoptosis, and Neoplastic Transformation: A Target for Cancer Chemotherapy. Leukemia 2003, 17, 590–603.
  9. Cain, R.J.; Ridley, A.J. Phosphoinositide 3-Kinases in Cell Migration. Biol. Cell 2009, 101, 13–29.
  10. Carnero, A.; Paramio, J.M. The PTEN/PI3K/AKT Pathway in Vivo, Cancer Mouse Models. Front. Oncol. 2014, 4, 252.
  11. Campbell, I.G.; Russell, S.E.; Choong, D.Y.H.; Montgomery, K.G.; Ciavarella, M.L.; Hooi, C.S.F.; Cristiano, B.E.; Pearson, R.B.; Phillips, W.A. Mutation of the PIK3CA Gene in Ovarian and Breast Cancer. Cancer Res. 2004, 64, 7678–7681.
  12. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.; Gazdar, A.; Powell, S.M.; Riggins, G.J.; et al. High Frequency of Mutations of the PIK3CA Gene in Human Cancers. Science 2004, 304, 554.
  13. Hartmann, C.; Bartels, G.; Gehlhaar, C.; Holtkamp, N.; von Deimling, A. PIK3CA Mutations in Glioblastoma Multiforme. Acta Neuropathol. 2005, 109, 639–642.
  14. Polom, K.; Marrelli, D.; Roviello, G.; Pascale, V.; Voglino, C.; Vindigni, C.; Generali, D.; Roviello, F. PIK3CA Mutation in Gastric Cancer and the Role of Microsatellite Instability Status in Mutations of Exons 9 and 20 of the PIK3CA Gene. Adv. Clin. Exp. Med. 2018, 27, 963–969.
  15. Wang, Y.; Wang, Y.; Li, J.; Li, J.; Che, G. Clinical Significance of PIK3CA Gene in Non-Small-Cell Lung Cancer: A Systematic Review and Meta-Analysis. BioMed Res. Int. 2020, 2020, 3608241.
  16. Rosty, C.; Young, J.P.; Walsh, M.D.; Clendenning, M.; Sanderson, K.; Walters, R.J.; Parry, S.; Jenkins, M.A.; Win, A.K.; Southey, M.C.; et al. PIK3CA Activating Mutation in Colorectal Carcinoma: Associations with Molecular Features and Survival. PLoS ONE 2013, 8, e65479.
  17. Arsenic, R.; Lehmann, A.; Budczies, J.; Koch, I.; Prinzler, J.; Kleine-Tebbe, A.; Schewe, C.; Loibl, S.; Dietel, M.; Denkert, C. Analysis of PIK3CA Mutations in Breast Cancer Subtypes. Appl. Immunohistochem. Mol. Morphol. 2014, 22, 50–56.
  18. Kalinsky, K.; Jacks, L.M.; Heguy, A.; Patil, S.; Drobnjak, M.; Bhanot, U.K.; Hedvat, C.V.; Traina, T.A.; Solit, D.; Gerald, W.; et al. PIK3CA Mutation Associates with Improved Outcome in Breast Cancer. Clin. Cancer Res. 2009, 15, 5049–5059.
  19. Martínez-Sáez, O.; Chic, N.; Pascual, T.; Adamo, B.; Vidal, M.; González-Farré, B.; Sanfeliu, E.; Schettini, F.; Conte, B.; Brasó-Maristany, F.; et al. Frequency and Spectrum of PIK3CA Somatic Mutations in Breast Cancer. Breast Cancer Res. 2020, 22, 45.
  20. Stemke-Hale, K.; Gonzalez-Angulo, A.M.; Lluch, A.; Neve, R.M.; Kuo, W.-L.; Davies, M.; Carey, M.; Hu, Z.; Guan, Y.; Sahin, A.; et al. An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer. Cancer Res. 2008, 68, 6084–6091.
  21. Kim, J.H. PIK3CA Mutations Matter for Cancer in Dogs. Res. Vet. Sci. 2020, 133, 39–41.
  22. Adams, J.R.; Xu, K.; Liu, J.C.; Agamez, N.M.R.; Loch, A.J.; Wong, R.G.; Wang, W.; Wright, K.L.; Lane, T.F.; Zacksenhaus, E.; et al. Cooperation between Pik3ca and P53 Mutations in Mouse Mammary Tumor Formation. Cancer Res. 2011, 71, 2706–2717.
  23. Koren, S.; Reavie, L.; Couto, J.P.; De Silva, D.; Stadler, M.B.; Roloff, T.; Britschgi, A.; Eichlisberger, T.; Kohler, H.; Aina, O.; et al. PIK3CA(H1047R) Induces Multipotency and Multi-Lineage Mammary Tumours. Nature 2015, 525, 114–118.
  24. Meyer, D.S.; Brinkhaus, H.; Müller, U.; Müller, M.; Cardiff, R.D.; Bentires-Alj, M. Luminal Expression of PIK3CA Mutant H1047R in the Mammary Gland Induces Heterogeneous Tumors. Cancer Res. 2011, 71, 4344–4351.
  25. Dong, L.; Meng, F.; Wu, L.; Mitchell, A.V.; Block, C.J.; Zhang, B.; Craig, D.B.; Jang, H.; Chen, W.; Yang, Q.; et al. Cooperative Oncogenic Effect and Cell Signaling Crosstalk of Co-occurring HER2 and Mutant PIK3CA in Mammary Epithelial Cells. Int. J. Oncol. 2017, 51, 1320–1330.
  26. Alsaihati, B.A.; Ho, K.-L.; Watson, J.; Feng, Y.; Wang, T.; Zhao, S. Canine Tumor Mutation Rate Is Positively Correlated with TP53 Mutation across Cancer Types and Breeds. bioRxiv 2020, 205286.
  27. Kim, T.-M.; Yang, I.S.; Seung, B.-J.; Lee, S.; Kim, D.; Ha, Y.-J.; Seo, M.-K.; Kim, K.-K.; Kim, H.S.; Cheong, J.-H.; et al. Cross-Species Oncogenic Signatures of Breast Cancer in Canine Mammary Tumors. Nat. Commun. 2020, 11, 3616.
  28. Lee, K.-H.; Hwang, H.-J.; Noh, H.J.; Shin, T.-J.; Cho, J.-Y. Somatic Mutation of PIK3CA (H1047R) Is a Common Driver Mutation Hotspot in Canine Mammary Tumors as Well as Human Breast Cancers. Cancers 2019, 11, 2006.
  29. Asproni, P.; Millanta, F.; Ressel, L.; Podestà, F.; Parisi, F.; Vannozzi, I.; Poli, A. An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors. Animals 2021, 11, 365.
  30. Ressel, L.; Millanta, F.; Caleri, E.; Innocenti, V.M.; Poli, A. Reduced PTEN Protein Expression and Its Prognostic Implications in Canine and Feline Mammary Tumors. Vet. Pathol. 2009, 46, 860–868.
  31. Pérez-Tenorio, G.; Alkhori, L.; Olsson, B.; Waltersson, M.A.; Nordenskjöld, B.; Rutqvist, L.E.; Skoog, L.; Stål, O. PIK3CA Mutations and PTEN Loss Correlate with Similar Prognostic Factors and Are Not Mutually Exclusive in Breast Cancer. Clin. Cancer Res. 2007, 13, 3577–3584.
  32. Maruyama, N.; Miyoshi, Y.; Taguchi, T.; Tamaki, Y.; Monden, M.; Noguchi, S. Clinicopathologic Analysis of Breast Cancers with PIK3CA Mutations in Japanese Women. Clin. Cancer Res. 2007, 13, 408.
  33. Mahajan, K.; Mahajan, N.P. PI3K-Independent AKT Activation in Cancers: A Treasure Trove for Novel Therapeutics. J. Cell Physiol. 2012, 227, 3178–3184.
  34. Arsenic, R.; Treue, D.; Lehmann, A.; Hummel, M.; Dietel, M.; Denkert, C.; Budczies, J. Comparison of Targeted Next-Generation Sequencing and Sanger Sequencing for the Detection of PIK3CA Mutations in Breast Cancer. BMC Clin. Pathol. 2015, 15, 20.
  35. Qiu, C.; Lin, D.; Wang, J.; Wang, L. Expression and Significance of PTEN in Canine Mammary Gland Tumours. Res. Vet. Sci. 2008, 85, 383–388.
  36. Milella, M.; Falcone, I.; Conciatori, F.; Cesta Incani, U.; Del Curatolo, A.; Inzerilli, N.; Nuzzo, C.M.A.; Vaccaro, V.; Vari, S.; Cognetti, F.; et al. PTEN: Multiple Functions in Human Malignant Tumors. Front. Oncol. 2015, 5, 24.
  37. Rasotto, R.; Berlato, D.; Goldschmidt, M.H.; Zappulli, V. Prognostic Significance of Canine Mammary Tumor Histologic Subtypes: An Observational Cohort Study of 229 Cases. Vet. Pathol. 2017, 54, 571–578.
  38. Riggio, M.; Perrone, M.C.; Polo, M.L.; Rodriguez, M.J.; May, M.; Abba, M.; Lanari, C.; Novaro, V. AKT1 and AKT2 Isoforms Play Distinct Roles during Breast Cancer Progression through the Regulation of Specific Downstream Proteins. Sci. Rep. 2017, 7, 44244.
  39. Mayer, I.A.; Abramson, V.G.; Formisano, L.; Balko, J.M.; Estrada, M.V.; Sanders, M.E.; Juric, D.; Solit, D.; Berger, M.F.; Won, H.H.; et al. A Phase Ib Study of Alpelisib (BYL719), a PI3Kα-Specific Inhibitor, with Letrozole in ER+/HER2− Metastatic Breast Cancer. Clin. Cancer Res. 2017, 23, 26–34.
  40. Port Louis, L.R.; Varshney, K.C.; Nair, M.G. An Immunohistochemical Study on the Expression of Sex Steroid Receptors in Canine Mammary Tumors. ISRN Vet. Sci. 2012, 2012, 378607.
  41. André, F.; Ciruelos, E.; Rubovszky, G.; Campone, M.; Loibl, S.; Rugo, H.S.; Iwata, H.; Conte, P.; Mayer, I.A.; Kaufman, B.; et al. Alpelisib for PIK3CA-Mutated, Hormone Receptor-Positive Advanced Breast Cancer. N. Engl. J. Med. 2019, 380, 1929–1940.
Video Production Service