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. 2022 Oct;86(4):286–293.

Coincidence of v-raf murine sarcoma viral oncogene homolog B mutation (V595E) with phosphorylated v-raf murine sarcoma viral oncogene homolog B in urothelial carcinoma in dogs

Hirofumi Yamasaki 1, Yosuke Uematsu 1, Yuhei Hayashi 1, Masao Yamashita 1, Meina Tei 1, Kazuyuki Uchida 1, Kenichiro Ono 1,✉, Hidehiro Hirao 1
PMCID: PMC9536355  PMID: 36211215

Abstract

Expression of phosphorylated v-raf murine sarcoma viral oncogene homolog B (pBRAF) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) were investigated in urothelial carcinoma (UC) in dogs with or without the BRAF gene mutation (V595E). Among the 10 cases of UC with V595E (−), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was reported in 8, with 7 displaying moderate reactivity and 1 displaying intense reactivity. Nuclear immunoreactivity against pBRAF was detected in 5 cases; however, these reactivities were non-specific, due to pBRAF being limited in the cytoplasm. In addition, positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 7 cases and nuclear immunoreactivity against ERK1/2 was detected in 6 cases. Among the 13 cases of UC with V595E (+), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pBRAF was detected in 10 cases; however, the nuclear immunoreactivity was non-specific. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pERK1/2 was also detected in all cases. As nuclear pERK1/2 indicates a progressive signaling process in the mitogen-activated protein kinase pathway, V595E (+) UC might be in its growing stage. Probable phosphorylated sites of pBRAF at Thr598/Ser601, detected in this study, are major and essential sites of the upstream rat sarcoma viral oncogene homolog (RAS) signaling pathway. In human cancers, the BRAF mutation never coincides with oncogenic RAS. To our knowledge, this is the first report on the simultaneous occurrence of the BRAF mutation (V595E) and pBRAF expression (at Thr598/Ser601) in dogs with UC with V595E (+).

Introduction

Urothelial carcinoma (UC), well-known as transitional cell carcinoma, is the most common tumor in the urinary tract, especially in the urinary bladder in dogs. The tumor, originating from urinary tract epithelium cells, shows highly invasive behavior into the lamina propria and distant metastasis, including the lymph node, lung, bone, muscle, and skin (1–3). A definitive diagnosis is fundamentally made by histopathologic analysis of surgically resected tumor tissues and/or endoscopic tissue samples. Recently, the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene mutation (V595E), corresponding to human V600E, was identified (4) and detected in various tumors in dogs (5), especially in those with UC, with high prevalence rates of 67 to 87% (4,6,7).

It is well-known that BRAF, one of the most important RAF family kinases, is a serine/threonine protein kinase that activates the mitogen-activated protein kinase (MAPK) signaling pathway. Activated BRAF, phosphorylated mainly by activated upstream the rat sarcoma viral oncogene homolog (RAS), and BRAF mutations phosphorylate related kinases, such as MAPK kinase (MEK) and its downstream extracellular signal-regulated kinase (ERK1/2), indicate oncogenicity and tumorigenicity in human cancers (8,9). Because over 90% of BRAF mutations are V600E, it is the most important and active oncogenic mutation (10–12). In canine UC, Decker et al (4) demonstrated that V595E induces the activation of the MAPK signaling pathway, including phosphorylated MEK and phosphorylated ERK1/2 (pERK1/2), and revealed its oncogenicity as the driver mutation of UC. In neoplastic cells, pERK1/2 (phospho-p44/42 MAPK) is translocated from the cytoplasm to the nucleus, which activates mitogenic factors (13,14).

This study investigates the expression of phosphorylated BRAF (pBRAF) and pERK1/2 in formalin-fixed tissue samples of UC in dogs with or without the BRAF mutation (V595E).

Materials and methods

Formalin-fixed tissue samples of UC in dogs

A total of 23 formalin-fixed and paraffin-embedded tissue samples of UC, none of which received treatment with BRAF inhibitors, were obtained from surgical cases at the Japan Animal Referral Medical Center and the Graduate School of Agricultural and Life Sciences Veterinary Medical Center at the University of Tokyo. An additional 4 cases of clinically healthy urinary bladder were obtained from necropsy cases from the Department of Veterinary Pathology at the University of Tokyo. All tissue samples were histopathologically confirmed as UC by 2 Board-certified veterinary pathologists at the Japanese College of Veterinary Pathologists. The UC samples were divided into 2 groups: with or without the BRAF mutation (V595E) detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Detection of BRAF mutation (V595E) by PCR-RFLP

Detection of V595E was performed according to previously reported PCR-RFLP method (4,6) with the original forward primer. Genomic DNA was extracted from 4-μm-thick sections for analysis of the BRAF gene mutation in exon 15 (V595E). Sections were deparaffinized by incubation with xylene at room temperature (RT) overnight. Xylene was removed in 100% ethanol at RT for 10 min. After centrifugation, ethanol was removed, and tissue pellets were dried up. The pellets were dissolved completely in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 1% SDS, pH 8.0) containing 100 ng/mL proteinase K at 70°C. The dissolving samples were added to an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v), mixed thoroughly and centrifuged to separate the aqueous layer. The aqueous layer containing the genomic DNA was transferred to a new tube, an equal volume of 2-propanol was added, and the mixture was centrifuged to precipitate genomic DNA. The genomic DNA was washed with 70% ethanol, dried, and dissolved with Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

The reverse primer, 5′-TGG CCT CAA TTC TTA CCA TCC AC-3′, designed by Decker et al (4) and the forward primer, 5′-GTA ATG CTT GCT TTG CTA GGA 3′, originally designed from genomic DNA, based on GenBank (accession No. NC_006598.2) were used for PCR. The PCR was performed using TaKaRa Ex Taq HS (Takara Bio, Shiga, Japan). The DNA sample (100 ng) was amplified in 50 μL of Tris-HCl buffer with 1.25 U TaKaRa Ex Taq HS, 200 μM dNTP, 2.0 mM of MgCl2, and 25 pM of each specific primer. The PCR conditions were 2 min at 94°C, followed by 35 (or 45) cycles of 20 s at 94°C, 20 s at 55°C, 20 s at 72°C, and finally, 10 min at 72°C for extension. The PCR products were detected by the 7.5% acrylamide gel electrophoresis and the target band was purified using LaboPassTM PCR (Hokkaido System Science, Sapporo, Hokkaido, Japan). Purified amplicons were submitted to the Fasmac (Atsugi, Kanagawa, Japan) for sequencing, using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, USA).

Using the primer pairs mentioned, a 196-base pair (bp) DNA fragment, corresponding to the human BRAF gene exon 15, was amplified. When V595E with a nucleotide substitution (c.1784T > A) corresponding to human V600E with a substitution of c.1799T > A was developed, the cleavage site of the BtsIMutl restriction enzyme (New England Biolabs Japan, Tokyo, Japan) sequencing CAGTG, disappeared (Figure 1 A, B). As PCR amplicons of the wild type (WT) BRAF gene were digested by BtsIMutI, V595E was clearly detected by PCR-RFLP analysis. The amplicon of the WT BRAF gene was digested into 2 DNA fragments of 122 and 74 bp, whereas the BRAF mutation (V595E) was not digested, demonstrating 3 fragments (Figure 1 C).

Figure 1.

Figure 1

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Genomic DNA sequence, PCR-sequencing chromatogram, and PCR-RFLP electropherogram of BRAF exon 15 gene with or without V595E [mutant (Mu) or wild type (WT)]. A — DNA sequence of WT and Mu BRAF gene in human (NM_004333.4) (upper line) and in dog (NC_006598.2) (lower line). The sequences are highly conserved between humans and dogs, although 2 silent point mutations are observed (green band). Using the primer pairs mentioned, a 196-base pair (bp) DNA fragment corresponding to human BRAF exon 15 gene is amplified and sequenced (from c1726 to c1845). Squared 5 genomic bases, in which the 3 latter bases consist of codon 595 and include the mutant sequence (red character), are the digestion site of BtsIMutI restriction enzyme. B — Chromatogram of the WT (left) and Mu (right) sequence of BRAF in dogs. When V595E with substituted c.1784T > A, corresponding to human V600E with substituted c.1799T > A is developed, the restriction enzyme site sequencing CAGTG disappears. C — V595E is clearly detected on the PCR-RFLP electropherogram. The amplicons are digested to 2 fragments of 122 and 74 bp in WT sequence, whereas the amplicon is not digested in the Mu sequence, demonstrating 3 bands of 196, 122, and 74 bp fragments.

Immunohistochemical detection of pBRAF and pERK1/2

Expression of pBRAF and pERK1/2 was immunohistochemically examined using 4-μm tissue sections of UC. Antigen retrieval was performed by autoclaving tissue sections in citrate buffer (pH 6.0) at 121°C for 10 min. Endogenous peroxidase activity in the tissue sections were inactivated with 3% hydrogen peroxide in methanol at RT for 5 min. To block nonspecific reactions, the sections were incubated with 8% skimmed milk in Tris-buffered saline (TBS) at RT for 30 min. The sections were then incubated at 4°C overnight with each primary antibody: rabbit polyclonal anti-BRAF (phosphor Thr598/Ser601) (Cat GTX85596, 1:100; Gene Tex, Irvine, California, USA) and rabbit monoclonal anti-phospho-p44/42 MAPK (ERK1/2) (Thr 202/Tyr204) (20G11) (Cat #4376S, 1:400; CST Japan, Tokyo, Japan). After 3 washings with TBS, the sections were treated with Dako EnVision+ System-horseradish peroxidase (HRP)-labeled polymer anti-rabbit secondary antibodies (Agilent Technologies Japan, Tokyo, Japan) at 37°C for 40 min. The chromogen consisted of 0.05% 3-3′-diaminobenzidine and 0.03% hydrogen peroxide in TBS. The sections were counterstained with Mayer’s hematoxylin. For the pBRAF and pERK1/2 negative controls, the primary antibodies were replaced with TBS. The images of cytoplasmic immunoactivity were tentatively classified as negative, weak positive, moderately positive, and intensely positive against pBRAF and pERK1/2.

Results

Detection of BRAF mutation (V595E) in formalin-fixed tissue of UC

In 13 out of 23 (57%) cases in this study, V595E mutation was detected in formalin-fixed tissue sections of UC. Although the prevalence rate of V595E was low, the results of the PCR-RFLP analysis provided a clear genomic DNA sequence and PCR sequencing chromatogram of V595E (Table I; Figure 1).

Table 1.

Expression of pBRAF and pERK1/2 in urothelial carcinoma with BRAF mutation (V595E) in dogs.

Immunoreactivity (cytoplasmic) Urothelial carcinoma Clinically healthy
V595E (+) (n = 13) V595E (−) (n = 10)
pBRAF
 negative (−) 2/10 (20%) 3/4 (80%)
 weak positive (±) 1/4 (20%)
 moderately positive (+) 2/13 (15%) 7/10 (70%)
 intensely positive (++) 11/13 (85%) 1/10 (10%)
pERK1/2
 negative (−) 3/10 (30%)
 weak positive (±)
 moderately positive (+) 1/13 (8%) 2/10 (20%)
 intensely positive (++) 12/13 (92%) 5/10 (50%)
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pBRAF: phosphorylated BRAF, pERK1/2: phosphorylated ERK1/2.

Expression of pBRAF and pERK1/2 in UC

Among the 4 cases of clinically healthy urinary bladder, weak positive cytoplasmic immunoreactivity against pBRAF was detected in the urothelial cell layer of 1 case. In the remaining 3 cases, neither cytoplasmic nor nuclear immunoreactivity against both pBRAF and pERK1/2 were observed (Figure 2).

Figure 2.

Figure 2

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Expression of pBRAF and pERK1/2 in 4 cases of clinically healthy urinary bladder of dogs. Weak positive cytoplasmic immunoreactivity against pBRAF is detected in the urothelial cell layer of 1 case (C 2). In the remaining 3 cases, neither cytoplasmic nor nuclear immunoreactivity against both pBRAF and pERK1/2 is observed (left columns: H&E staining, bar = 500 nm; center columns: pBRAF; right columns: pERK1/2). Negative controls are represented in the bottom row.

All 13 cases of UC with V595E (+) revealed moderately or intensely positive cytoplasmic immunoreactivity against both pBRAF and pERK1/2, whereas 1 out of 10 cases with V595E (−) showed intensely positive immunoreactivity against pBRAF and 5 out of 10 cases showed intensely positive immunoreactivity against pERK1/2 (Table I).

Among the 10 cases of UC with V595E (−), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was seen in 8 cases (7 moderately and 1 intensely). In the remaining 2 cases, there was no significant cytoplasmic immunoreactivity for pBRAF. Nuclear immunoreactivity against pBRAF was detected in 5 cases; however, these reactivities were non-specific, due to pBRAF being limited in cytoplasm. Positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 7 cases (2 moderately and 5 intensely). Nuclear immunoreactivity against ERK1/2 was detected in 6 cases (Figure 3).

Figure 3.

Figure 3

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Expression of pBRAF and pERK1/2 in canine UC with V595E (−). Every case of UC with V595E (−) shows some typical histopathological features of UC (left columns, H&E staining). In the case of WT 2, neoplastic foci consisting of the solid proliferation of large pleomorphic tumor cells are formed in the lamina propria (bar = 500 nm). In the case of WT 5, the tumor cells formed papillary structures toward the lumen and glandular structures in the lamina propria (bar = 500 nm). In the case of WT 10, neoplastic foci consisting of the solid proliferation of large pleomorphic tumor cells are formed in the lamina propria (bar = 500 nm). The results of immunohistochemistry against pBRAF are represented in the center columns and those against pERK1/2 are represented in the right columns. Among 10 cases of UC with V595E (−), cytoplasmic immunoreactivity against pBRAF of neoplastic cells is seen in 8 cases (7 moderately: WT 1–3, 6–9; 1 intensely: WT 5). In the remaining 2 cases (WT 4 and 10), there is no significant cytoplasmic immunoreactivity for pBRAF. Nuclear immunoreactivity against pBRAF is detected in 5 cases (WT 2, 3, 5, 8, and 9); however, these reactivities are non–specific, due to pBRAF being limited in the cytoplasm. Positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells is detected in 7 cases (2 moderately: WT 6 and 7; 5 intensely: WT 3, 5, 8–10). Nuclear immunoreactivity against ERK1/2 is detected in 6 cases (WT 3, 5, 7, 8–10).

Among the 13 cases of UC with V595E (+), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was detected in 13 cases (2 moderately and 11 intensely). Nuclear immunoreactivity against pBRAF was detected in 10 cases; however, these reactivities were non-specific, like in V595E (−) cases. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 13 cases (1 moderately and 12 intensely). Nuclear immunoreactivity against pERK1/2 was detected in all 13 cases (Figure 4).

Figure 4.

Figure 4

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Expression of pBRAF and pERK1/2 in canine UC with V595E (+). Every UC with V595E (+) shows typical histopathological features of UC (left columns, H&E staining), especially in Mu 1, Mu 2, and Mu 3, where the neoplastic cells formed papillary structures toward the lumen and glandular structures in the lamina propria (bar = 500 nm). In the case of Mu 8, neoplastic foci consisting of pleomorphic tumor cells formed glandular structures in the lamina propria (bar = 500 nm). In the case of Mu 12, eosinophilic atypical neoplastic cells with anisocytosis and anisokaryosis proliferated (bar = 300 nm). The results of immunochemistry against pBRAF are represented in the center columns and those against pERK1/2 are represented in the right columns. Cytoplasmic immunoreactivity against pBRAF of neoplastic cells is detected in all 13 cases of UC with V595E (+) (2 moderately: Mu 7 and 12; 11 intensely: Mu 1–6, 8–11, 13). Nuclear immunoreactivity against pBRAF is detected in 10 cases; however, these reactivities are nonspecific, like in V595E (−) cases. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells is detected in 13 cases (1 moderately: Mu 7; 12 intensely: Mu 1–6, 8–13). Nuclear immunoreactivity against pERK1/2 is detected in all 13 cases.

Discussion

Although the incidence of UC is less than 2% of all malignant tumors in dogs, almost all of them (97%) are invasive malignant tumors (1,15). The tumor is quite similar in terms of histopathology, biological behavior, response to chemotherapy, and prognosis to human invasive UC (15–18). Various risk factors are reported in canine UC, including age of onset (over 10-years-old: 45%), sex predilection (female: twice as likely as a male), breed-associated predisposition (Scottish terrier: 21-fold increased risk), major histological features with a high grade papillary infiltrative tumor, high incidence of local and distant metastases (49%), and poor prognosis (median survival time of post-surgery: 125 d) (1,19,20). In addition, incidence rates of V595E mutation are reported in 67 to 87% of UC in dogs (4,5,7). In this study, a low prevalence rate of V595E (57%; 13/23 UC cases) was detected. Some risk factors, such as sex predilection (female), breed population (high risk in terrier breed), and size of samples (50, 67, and 80% in 16, 45, and 66 samples, respectively) (4–6), might be related to the low prevalence of V595E mutation in this study.

It is widely accepted that the MAPK signaling pathway consists of the ERK1/2 pathway, the regulating oncogenic pathway, and the Jun N-terminal kinase (JNK1/2/3) and P38 (P38α/β/γ/δ) pathway, regulating inflammation and the stress response (21,22). In the ERK1/2 pathway, the receptor tyrosine kinase (RTK)-rat sarcoma viral oncogene homolog (RAS)-RAF-MEK-ERK cascade, RTK-RAS-phosphatidylinositol-3-kinase-mammalian target of rapamycin cascade, or G protein coupled receptor-guanine nucleotide-binding protein (GNAG)-MEK-ERK1/2 cascade is closely related to the oncogenic driver in cancer (23–25). Especially the RTK-RAS-RAF-MEK-ERK1/2 cascade, which is the most important cascade for tumorigenesis (26,27).

All 13 cases of UC with V595E (+) revealed moderately or intensely positive immunoreactivity against pERK1/2, indicating activation of the MAPK signaling pathway. The V595E, like other BRAF mutations, can directly phosphorylate and activate MEK with subsequent activation of ERK1/2 by changing the conformation of the BRAF activating loop (28,29). In this study, immunoreactivity against pERK1/2, reflecting phosphor-p44/42 MAPK (ERK1/2), was detected in the cytoplasm and nucleus of neoplastic cells. In addition, 11 out of 13 cases of UC with V595E (+) revealed nuclear immunoreactivity against pERK1/2. Since nuclear pERK1/2 translocated from the cytoplasm indicates the progressive signaling process in the MAPK pathway (13,14), V595E (+) UC might be the growing stage of the tumor.

On the other hand, cytoplasmic immunoreactivity against pBRAF, indicating phosphor Thr598/Ser601 BRAF, was detected in all 13 cases of UC with V595E (+), with moderately or intensely positive reactivity, like those with pERK1/2 immunoreactivity. As pBRAF is fundamentally limited in cytoplasm (8,30), reactivity against nuclear pBRAF, observed in neoplastic cells of some cases in this study, is non-specific, except for inflammatory cells in WT 2. Phosphorylation sites of BRAF at Thr598/Ser601 are major and essential sites for activating upstream RAS signaling, consisting of the RKT-RAS-BRAF-MEK-ERK cascade (31,32). Whereas UC with V595E (−) showed negative to intensely positive immunoreactivity, probably because some of them had an activated RAS or GNAG (31–34). Interestingly, the BRAF gene mutation (V600E) never coincides with activated RAS, including the KRAS or HRAS mutations, resulting in pBRAF (phosphor Thr598/Ser601) in human cancers (12,35,36). In contrast, many researchers report that pBRAF is detected in melanoma cases with acquired resistance to BRAF inhibitors caused by the paradoxical activation of upstream signaling RTK or RAS through the ERK1/2 feedback mechanism (37–40).

To our knowledge, this is the first report on the BRAF mutation (V595E) coinciding with pBRAF expression (at Thr598/Ser601). Further studies are necessary to elucidate the pathophysiological meaning of pBRAF in canine UC with V595E (+).

References

  • 1.Knapp DW, Glickman NW, Denicola DB, Bonney PL, Lin TL, Glickman LT. Naturally-occurring canine transitional cell carcinoma of the urinary bladder: A relevant model of human invasive bladder cancer. Urol Oncol. 2000;5:47–59. doi: 10.1016/s1078-1439(99)00006-x. [DOI] [PubMed] [Google Scholar]
  • 2.Knapp DW, Ramos-Vara JA, Moore GE, Dhawan D, Bonney PL, Young KE. Urinary bladder cancer in dogs, a naturally occurring model for cancer biology and drug development. ILAR J. 2014;55:100–118. doi: 10.1093/ilar/ilu018. [DOI] [PubMed] [Google Scholar]
  • 3.Reed LT, Knapp DW, Miller MA. Cutaneous metastasis of transitional cell carcinoma in 12 dogs. Vet Pathol. 2013;50:676–681. doi: 10.1177/0300985812465326. [DOI] [PubMed] [Google Scholar]
  • 4.Decker B, Parker HG, Dhawan D, et al. Homologous mutation to human BRAF V600E is common in naturally occurring canine bladder cancer – Evidence for a relevant model system and urine-based diagnostic test. Mol Cancer Res. 2015;13:993–1002. doi: 10.1158/1541-7786.MCR-14-0689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mochizuki H, Kennedy K, Shapiro SG, Breen M. BRAF mutations in canine cancers. PLoS One. 2015;10:e0129534. doi: 10.1371/journal.pone.0129534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Mochizuki H, Shapiro SG, Breen M. Detection of BRAF mutation in urine DNA as a molecular diagnostic for canine urothelial and prostatic carcinoma. PLoS One. 2015;10:e0144170. doi: 10.1371/journal.pone.0144170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Aupperle-Lellbach H, Grassinger J, Hohloch C, Kehl A, Pantke P. Diagnostic value of the BRAF variant V595E in urine samples, smears and biopsies from canine transitional cell carcinoma. Tierarzt Prax Ausg K Kleintiere Heimtiere. 2018;46:289–295. doi: 10.15654/TPK-180554. [DOI] [PubMed] [Google Scholar]
  • 8.Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–954. doi: 10.1038/nature00766. [DOI] [PubMed] [Google Scholar]
  • 9.Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116:855–867. doi: 10.1016/s0092-8674(04)00215-6. [DOI] [PubMed] [Google Scholar]
  • 10.Ascierto PA, Kirkwood JM, Grob JJ, et al. The role of BRAF V600 mutation in melanoma. J Transl Med. 2012;10:85. doi: 10.1186/1479-5876-10-85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Dhillon AS, Kolch W. Oncogenic B-Raf mutations: Crystal clear at last. Cancer Cell. 2004;5:303–304. doi: 10.1016/s1535-6108(04)00087-x. [DOI] [PubMed] [Google Scholar]
  • 12.Haling JR, Sudhamsu J, Yen I, et al. Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling. Cancer Cell. 2014;26:402–413. doi: 10.1016/j.ccr.2014.07.007. [DOI] [PubMed] [Google Scholar]
  • 13.Maik-Rachline G, Hacohen-Lev-Ran A, Seger R. Nuclear ERK: Mechanism of translocation, substrates, and role in cancer. Int J Mol Sci. 2019;20:1194. doi: 10.3390/ijms20051194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Parikh N, Shuck RL, Nguyen TA, Herron A, Donehower LA. Mouse tissues that undergo neoplastic progression after K-Ras activation are distinguished by nuclear translocation of phospho-Erk1/2 and robust tumor suppressor responses. Mol Cancer Res. 2012;10:845–855. doi: 10.1158/1541-7786.MCR-12-0089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Norris AM, Laing EJ, Valli VE, et al. Canine bladder and urethral tumors: A retrospective study of 115 cases (1980–1985) J Vet Intern Med. 1992;6:145–153. doi: 10.1111/j.1939-1676.1992.tb00330.x. [DOI] [PubMed] [Google Scholar]
  • 16.Dhawan D, Paoloni M, Shukradas S, et al. Comparative gene expression analyses identify luminal and basal subtypes of canine invasive urothelial carcinoma that mimic patterns in human invasive bladder cancer. PLoS One. 2015;10:e0136688. doi: 10.1371/journal.pone.0136688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dhawan D, Ramos-Vara JA, Stewart JC, Zheng R, Knapp DW. Canine invasive transitional cell carcinoma cell lines: In vitro tools to complement a relevant animal model of invasive urinary bladder cancer. Urol Oncol. 2009;27:284–292. doi: 10.1016/j.urolonc.2008.02.015. [DOI] [PubMed] [Google Scholar]
  • 18.Patrick DJ, Fitzgerald SD, Sesterhenn IA, Davis CJ, Kiupel M. Classification of canine urinary bladder urothelial tumours based on the World Health Organization/International Society of Urological Pathology consensus classification. J Comp Pathol. 2006;135:190–199. doi: 10.1016/j.jcpa.2006.07.002. [DOI] [PubMed] [Google Scholar]
  • 19.Mutsaers AJ, Widmer WR, Knapp DW. Canine transitional cell carcinoma. J Vet Intern Med. 2003;17:136–144. doi: 10.1892/0891-6640(2003)017<0136:ctcc>2.3.co;2. [DOI] [PubMed] [Google Scholar]
  • 20.Nolan MW, Gieger TL, Vaden SL. Management of transitional cell carcinoma of the urinary bladder in dogs: Important challenges to consider. Vet J. 2015;205:126–127. doi: 10.1016/j.tvjl.2015.03.022. [DOI] [PubMed] [Google Scholar]
  • 21.Fulkerson CM, Dhawan D, Ratliff TL, Hahn NM, Knapp DW. Naturally occurring canine invasive urinary bladder cancer: A complementary animal model to improve the success rate in human clinical trials of new cancer drugs. Int J Genomics. 2017;2017:6589529. doi: 10.1155/2017/6589529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signaling pathways in cancer. Oncogene. 2007;26:3279–3290. doi: 10.1038/sj.onc.1210421. [DOI] [PubMed] [Google Scholar]
  • 23.Lee S, Rauch J, Kolch W. Targeting MAPK signaling in cancer: Mechanisms of drug resistance and sensitivity. Int J Mol Sci. 2020;21:1102. doi: 10.3390/ijms21031102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Porta C, Paglino C, Mosca A. Targeting PI3K/Akt/mTOR signaling in cancer. Front Oncol. 2014;4:64. doi: 10.3389/fonc.2014.00064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Thapa D, Stoner MW, Zhang M, et al. Adropin regulates pyruvate dehydrogenase in cardiac cell via novel GPCR-MAPK-PDK4 signaling pathway. Redox Biol. 2018;18:25–32. doi: 10.1016/j.redox.2018.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Santarpia L, Lippman SL, El-Naggar AK. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16:103–119. doi: 10.1517/14728222.2011.645805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Corcoran RB, Settleman J, Engelman JA. Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers. Oncotarget. 2011;2:336–346. doi: 10.18632/oncotarget.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Liu T, Wang Z, Guo P, Ding N. Electrostatic mechanism of V600E mutation-induced B-Raf constitutive activation in colorectal cancer: Molecular implications for the selectivity difference between type-I and type-II inhibitors. Eur Biophys J. 2019;48:73–82. doi: 10.1007/s00249-018-1334-y. [DOI] [PubMed] [Google Scholar]
  • 29.Maloney RC, Zhang M, Jang H, Nussinov R. The mechanism of activation of monomeric B-Raf V600E. Comput Struct Biotechnol J. 2021;19:3349–3363. doi: 10.1016/j.csbj.2021.06.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Loo E, Khalili P, Beuhler K, Siddiqi I, Vasef MA. BRAF V600E mutation across multiple tumor types: Correlation between DNA-based sequencing and mutation-specific immunohistochemistry. Appl Immunohistochem Mol Morphol. 2018;26:709–713. doi: 10.1097/PAI.0000000000000516. [DOI] [PubMed] [Google Scholar]
  • 31.Zhang BH, Guan KL. Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601. EMBO J. 2000;19:5429–5439. doi: 10.1093/emboj/19.20.5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zhang BH, Guan KL. Regulation of the Raf kinase by phosphorylation. Exp Lung Res. 2001;27:269–295. doi: 10.1080/019021401300054046. [DOI] [PubMed] [Google Scholar]
  • 33.Eisenhardt AE, Olbrich H, Röring M, et al. Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma. Int J Cancer. 2011;129:2297–2303. doi: 10.1002/ijc.25893. [DOI] [PubMed] [Google Scholar]
  • 34.Chong H, Lee J, Guan KL. Positive and negative regulation of Raf kinase activity and function by phosphorylation. EMBO J. 2001;20:3716–3727. doi: 10.1093/emboj/20.14.3716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Dvořák K, Higgins A, Palting J, Cohen M, Brunhoeber P. Immunohistochemistry with anti-BRAF V600E (VE1) mouse monoclonal antibody is a sensitive method for detection of the BRAF V600E mutation in colon cancer: Evaluation of 120 cases with and without KRAS mutation and literature review. Pathol Oncol Res. 2019;25:349–359. doi: 10.1007/s12253-017-0344-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Mochizuki H, Breen M. Sequence analysis of RAS and RAF mutation hot spots in canine carcinoma. Vet Comp Oncol. 2017;15:1598–1605. doi: 10.1111/vco.12275. [DOI] [PubMed] [Google Scholar]
  • 37.Little AS, Balmanno K, Sale MJ, et al. Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells. Sci Signal. 2011;4:ra17. doi: 10.1126/scisignal.2001752. [DOI] [PubMed] [Google Scholar]
  • 38.Holderfield M, Merritt H, Chan J, et al. RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation. Cancer Cell. 2013;23:594–602. doi: 10.1016/j.ccr.2013.03.033. [DOI] [PubMed] [Google Scholar]
  • 39.Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010;468:973–977. doi: 10.1038/nature09626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Prahallad A, Sun C, Huang S, et al. Unresponsiveness of colon cancer to BRAF (V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100–103. doi: 10.1038/nature10868. [DOI] [PubMed] [Google Scholar]

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