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F1000Research Mutations in the PIK3C2B , ERBB3 , KIT, and MLH1 Genes and their Relationship with Resistance to Temozolomide in Patients with High-grade Gliomas León Darío Ortiz Gómez1,2, Heidy Johanna Contreras Martínez^3 , David Andrés Galvis Pareja^3 , and Ronald Guillermo Peláez Sánchez^4 (^1) Doctoral Program in Health Sciences, Graduate School, CES University, Medellín, Colombia. (^2) Cancer Institute, Las Americas-AUNA Clinic, Medellín, Colombia. (^3) Pharmaceutical Sciences Research Group (ICIF), CES University, Medellín, Colombia. (^4) Life and Health Sciences Research Group, Graduate School, CES University, Medellín, Colombia. Runing title: Mutations in high-grade gliomas *** Correspondence:* Ronald Guillermo Peláez Sánchez, [email protected], Phone: (57) (604) 4440555 Extension 1750 Introduction. The treatment for patients with high-grade gliomas includes radiation therapy and temozolomide. However, some patients do not respond to temozolomide because they have a methylation reversal mechanism through the enzyme O6-methylguanine-DNA-methyltransferase. This biomarker has been used as a prognostic factor in patients receiving treatment with temozolomide. However, not all patients respond in the same way, which suggests the existence of other genes involved in resistance to temozolomide. Methods. A group of 31 patients with high- grade gliomas was recruited and were clinically, image pattern, and pathologically characterized. The sequencing of 324 genes related to different types of cancer was performed to detect mutations. Subsequently, a statistical analysis was conducted to determine the mutated genes that were most related to resistance to treatment. Results. The genes related to the second relapse of patients with high-grade glioma after the use of temozolomide according to Stupp protocol and metronomic dose were PIK3C2B, KIT, ERBB3, and MLH1. Conclusion. Considering the results obtained, we suggest that the mutations in the four genes and the methylation of the gene promoter that codes for MGMT protein could be related to the clinical evolution of patients with high-grade gliomas and its response to treatment with temozolomide. Keywords: gliomas, temozolomide, resistance, genes, PIK3C2B , ERBB3 , KIT , MLH1 , MGMT. 1 1 1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 2
INTRODUCTION
According to the data provided by Cancer Today 2020, the incidence of brain tumors worldwide is 3.5 per 100,000 inhabitants, with a mortality rate of 2.8 per 100,000 inhabitants (1). The incidence (standardized rate) of cancer in the brain and central nervous system in Colombia ranged from 3.5– 4.2 per 100,000 inhabitants, according to data obtained between 2003 and 2016. While mortality ranged between 2.1 and 2.5 per 100,000 inhabitants between 1997 and 2020 (https://infocancer.co/) (2), according to the Brain Tumor Registry (CBTRUS, 2014–2018), the incidence in the United States for primary central nervous system (CNS) tumors was 24 per 100,000, with a mortality rate of 4.3 per 100,000 (3). Concerning the cases reported in the United States, these data reflect an underreporting of new cases in the rest of the world, including Colombia. Additionally, the CBTRUS estimates that for the United States, the incidence of gliomas, in general, is 5.95, and for the subgroup considered high-grade gliomas, that is, glioblastoma, it is 3.23, anaplastic astrocytoma is 0.41, and anaplastic oligodendroglioma is 0.11 per 100,000 inhabitants (3). The most abundant cells in the CNS are neurons and glial cells. The glial cells are composed of astrocytes, oligodendrocytes, ependymal cells, and microglia cells. Additionally, meningeal, and pituitary cells are also part of the CNS (4). When cells have genetic and/or epigenetic alterations, mechanisms such as replication, repair, and senescence are modified, resulting in an uncontrolled proliferation of cells with aggressive characteristics that allow them to invade neighboring tissues and sometimes give rise to metastasis, even in isolated and immune privileged sites such as the brain parenchyma (5). Tumors originating from astrocytes are called astrocytomas; tumors from oligodendrocytes are oligodendrogliomas; and tumors from ependymal cells are called ependymomas. Additionally, these types of malignant brain tumors are classified according to their degree of differentiation into low-grade and high-grade (6). High-grade gliomas, now named according to the 2021 WHO classification of tumors of the central nervous system, include IDH wild-type glioblastoma (GBwt-IDH), IDH-mutated astrocytoma without 1p/19q codeletion, grades 3 and 4 (Amut-IDH, sin-codel-1p/19q, G3-4), and oligodendroglioma mutated for IDH with codeletion 1p/19q grade 3 (Omut-IDH, codel-1p/19q, G3)^ (7). Regarding high-grade glioma treatment, the protocol developed by Stupp et al. (2005) is used as standard treatment. This protocol added temozolomide to treatment with radiotherapy, reporting an increase in patient survival (HR not adjusted: 0.63, 95% CI: 0.52–0.75, p < 0.001) and minimal toxicity (8). However, the treatment was not effective for all patients, so it was necessary to explore at the genomic level which gene was related to resistance to temozolomide (9), finding that promoter methylation of the gene that codes for the MGMT protein had an independent prognostic value of treatment (HR: 0.45, 95%, CI: 0.32– 0.61, p < 0.001) and decreased the probability of death (HR: 0.51, 95% CI: 0.31–0.84) in patients in whom temozolomide was added to radiotherapy, the same did not occur in patients who did not have the gene promoter methylated (HR: 0.69, 95% CI: 0.47–1.02) (10), which indicates that this mechanism of resistance to temozolomide does not explain the response to treatment in 100% of the patients. Accordingly, there is a gap in our knowledge about other mechanisms or proteins that are involved in the process of resistance to treatment with temozolomide in patients with high-grade gliomas. Therefore, the objective of our investigation was to identify additional genetic alterations to the promoter's methylation of the gene that codes for the MGMT protein, which helps relate the clinical evolution of patients with high-grade gliomas and their response to treatment with temozolomide. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
Bioinformatic analysis of mutations effect on the proteins The chemical properties of the proteins were identified using the ProtParam tool web server (https://web.expasy.org/protparam/). The identification of the chemical properties of the amino acids was conducted by means of the GPMAW web server (https://www.alphalyse.com/customer- support/gpmaw-lite-bioinformatics-tool/start-gpmaw-lite/) and the color protein sequence web server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_color.html). The effect of the mutations on the proteins was determined by the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.bii.a-star.edu.sg/) web servers. The signal peptide of the proteins was identified using the SignalP-6 web server (https://dtu.biolib.com/SignalP-6). The InterProScan web server was used to identify the domains, biological processes, molecular function, and cellular component of the four proteins (https://www.ebi.ac.uk/interpro). Subsequently, using the String Interaction Network web server (STRING) and Genecards database, the interactions, and metabolic pathways in which the proteins participate were identified (https://string-db.org/ and https://www.genecards.org). The three- dimensional structure of the proteins was modeled using the Swiss-Model web server (https://swissmodel.expasy.org), and the structural change caused by the mutations was determined using the Swiss-pdb-viewer bioinformatics program (http://spdbv.unil.ch), to establish how the structure and function of the protein were affected. Finally, all the diseases that have been associated with mutations in the four genes were identified using the OMIM database (https://www.omim.org). Results Study population A total of 31 patients, who met the inclusion criteria and agreed to enter the study by signing the informed consent, participated in the present study. In relation to the clinical data, the mean age at diagnosis of high-grade glioma was 47 years old (SD 14.52). Regarding gender, 11 patients were male (35.49%) and 20 were female (64.51%). Clinical information The Karnofsky scale was applied to the 31 patients included in the study to assess their functional status at the time of diagnosis, finding values equal to or greater than 70% in all patients, which indicated that all were fit to be taken to surgery. All patients underwent contrasted preoperative magnetic resonance imaging of the brain, which was recorded in the patients' medical records. Without discriminating by which cerebral hemisphere was injured and considering that sometimes the lesions are not confined to a single lobe, it was found that 32% of the patients had the lesion in the frontal lobe, 16% in the parietal, 22% in the temporal, 26% in the occipital, and only one patient had a lesion in the basal ganglia. A grossly complete resection was performed on 45% of the patients, and a partial resection or biopsy was performed on the remainder (55%). According to the pathology report, based on hematoxylin-eosin (H-E) and immunohistochemistry (IHC), it was found that 21 (68%) of the patients had glioblastomas, five (16%) had anaplastic astrocytomas, and five (16%) had oligodendroglial lineage tumors. The latter group was made up of four patients with anaplastic oligoastrocytomas and one patient with an anaplastic oligodendroglioma; see Figure 1. 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160
All patients were given concomitant treatment with temozolomide and radiotherapy, followed by adjuvant chemotherapy four to six weeks after surgery. In 15 patients, it was possible to prevent tumor growth with six cycles of temozolomide as recommended by the Stupp Protocol (SP). However, five patients received fewer cycles due to clinical deterioration and tumor growth (progression). While in the remaining 11 patients it was necessary to administer more than six cycles of temozolomide to prevent tumor growth; see Figure 2. Patients were followed clinically and with imaging, until tumor growth was observed again (first relapse). All patients were given metronomic temozolomide; that is, a dose of 75 mg/mt2 every day to try to avoid the effect of MGMT as this enzyme has a very short half-life. The response of patients to treatment with metronomic temozolomide was variable, ranging from patients who reached the second relapse in the second treatment cycle to patients who reached the second relapse in the 21 cycles of temozolomide treatment at metronomic doses; see Figure 3. Sample collection for molecular biology analysis To classify high-grade gliomas, the following molecular analyses were performed on the samples obtained from the first surgery: a search of the 1p/19q codeletion by FISH in three patients (10% of the sample), which helped to classify them as ana-plastic oligoastrocytoma. Immunohistochemistry (IHC) for IDH1 was performed on 19 patients (61%), reporting positive results in 4/10 glioblastomas (40%), 3/4 ana-plastic astrocytomas (75%), and in 5/5 oligodendroglial tumors (100%). The methylation status of the promoter of the gene that codes for the MGMT protein was searched by polymerase chain reaction (PCR) in 18 patients, finding methylation in 3/10 glioblastomas (30%), 2/3 anaplastic astrocytomas (66%), and 4/4 tumors of the oligodendroglial lineage (100%); see Supplementary Material 1. Sequencing 324 cancer-associated genes The genetic material was extracted from the 31 samples, and 324 cancer-related genes were sequenced and analyzed according to the protocol described by the FoundationOne®CDx (F1CDx) and FoundationONE Liquid CDx (F1LCDx) panels. An amplification, sequencing, and mutation detection was conducted on each of the genes. According to the results obtained in the gene profiling of the 31 patients with high-grade gliomas, 370 types of mutations were found in 185 genes. The ten most frequently mutated genes, in order of frequency, for the 21 patients initially diagnosed with glioblastomas were EGFR (62%), TERT (62%), PDGFA (57%), TP53 43%, CDKN2A/B 29%), NF (24%), ALK (19%), EP300 (19%), KDR (19%), and MLL2 (19%). The most frequently mutated genes in the five patients diagnosed with anaplastic astrocytomas were IDH1 (40%), TP53 (40%), CCN2 (40%), KEAP1 (40%), KIT 40%, MAF (40%), PDGFRA (40%), SPEN (40%), TERT 40%), and TYRO3 (40%). The most frequently mutated genes in the group of five patients with anaplastic oligoastrocytomas were TSC1 (75%), SPEN (50%), IDH (25%), ALK (25%), ARID1A (25%), CIC (25%), MAP3K1 (25%), MLL2 (25%), NF1 (25%), and POLE (25%); see supplementary material 2. Mutated genes identification related with temozolomide resistance mechanisms The simple Cox's regression analysis presents the comparison between the de-pendent variable "second relapse" (at 22 months) and the presence of mutated genes. Seventy-four mutated genes were found in patients who presented a second relapse. The genes with the greatest statistical significance found were PIK3C2B HR 13.81 (95%, CI: 2.25–84.45, p = 0.004), NOTCH3 HR 6.02 (95%, CI: 1.74–20.84, p = 0.005), KIT HR 3.98 (95%, CI: 1.20–13.18, p = 0.024), ERBB3 HR 3.87 (95%, CI: 1.06–14.04, p = 0.04), and MLH1 HR 3.52 (95%, CI: 0.95–13.09, p = 0.06); see Table 1. 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
to be 46.22 and classified as unstable. The R458Q mutation was detected in the protein. The wild- type amino acid is an arginine (R) that is classified as a basic, which is replaced by a glutamine (Q) that is hydrophilic. According to the results of PolyPhen-2 (prediction of functional effects of human nsSNPs), this mutation is predicted to be probably dam-aging with a score of 0.979 (sensitivity: 0.76; specificity: 0.96). According to the results of SIFT (Sorting Intolerant From Tolerant), substitution at position 458 from R to Q is predicted to be tolerated with a score of 0.45. No signal peptide was detected using the SignalP bioinformatics program (version 6.0). The effect of the mutation on the structure of the protein leads to the shortening of the radical, reducing the Van der Waals forces, which possibly affects the attraction between atoms, molecules, and surfaces. At the structural level, the mutation is located between the amino acids (457–464) that make up a beta sheet, generating an elongation of the beta sheet in the mutated protein. All these alterations can lead to instability and loss of functionality of the protein; see Table 5. The ERBB3 protein is made up of 1342 amino acids, has a molecular weight of 148098.19 Da, a theoretical isoelectric point of 6.11, 154 negatively charged residues (Asp + Glu), 132 positively charged residues (Arg + Lys), an atomic composition of 6447 carbon, 10126 hydrogen, 1852 nitrogen, 1969 oxygen, and 94 sulfur atoms, an estimated half-life of 30 hours (mammalian reticulocytes, in vitro).Its instability index is computed to be 49.61 and classifies as unstable. The R164K mutation was detected in the protein. The wild-type amino acid is an arginine (R) that is classified as a basic, which is replaced by a lysine (K) that is hydrophilic. According to the results of PolyPhen-2, this mutation is predicted to be probably benign with a score of 0.017 (sensitivity: 0.95; specificity: 0.80). According to the results of SIFT substitution at position 164 from R to K, it is predicted to be tolerated with a score of 0.64. No signal peptide was detected using the SignalP bioinformatics program (version 6.0). At the structural level, an amino acid change is observed that leads to an alteration in the orientation of the radical group that alters the spatial distribution of Van der Waals forces, which possibly affects the attractions be-tween atoms, molecules, and surfaces; see Table 5. The KIT protein is made up of 977 amino acids, has a molecular weight of 109992.70 Da, a theoretical isoelectric point of 6.54, 111 negatively charged residues (Asp + Glu), 106 positively charged residues (Arg + Lys), an atomic composition of 4925 carbon, 7657 hydrogen, 1301n, 1460 oxygen, and 48 sulfur atoms, and an estimated half-life of 30 hours (mammalian reticulocytes, in vitro). Its instability index is computed to be 39.43 and classified as stable. No signal peptide was detected using the SignalP bioinformatics program (version 6.0). This protein did not present a structural alteration but did show an increase in the number of copies of the KIT gene in the patient's genome. This genetic phenomenon is common in cancer cells, which produce several copies of one or more genes in response to signals from other cells or the environment. The MLH1 protein is made up of 756 amino acids, has a molecular weight of 84600 Da, a theoretical isoelectric point of 5.51, 104 negatively charged residues (Asp + Glu), 83 positively charged residues (Arg + Lys), an atomic composition of 3740 carbon, 5947 hydrogen, 1017 nitrogen, 1165 oxygen, and 25 sulfur atoms, and an estimated half-life of 30 hours (mammalian reticulocytes, in vitro). Its instability index is computed to be 51.29 and classified as unstable. The F261fs31 mutation was detected in the protein. No signal peptide was detected using the SignalP bioinformatics program (version 6.0). The deletion of a cytokine at position 783 of MLH1 gene (783delC), leads to structural change in the MLH1 protein (p.Phe261fs7). This sequence change creates a premature translational stop signal of 7 amino acids after phenylalanine (Frameshift). It is expected to result in an absent or disrupted protein product (this could lead to the production of a truncated protein of 268 amino acids); see Table 5. 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293
Finally, the diseases associated with each of the four mutated genes are identified using the OMIM database (An Online Catalog of Human Genes and Genetic Disorders). Mutations in the gene that codes for the PIK3C2B protein have been associated with the following diseases: high-grade glioma, Maffucci, and Hepatitis C. Mutations in the gene that codes for ERBB3 protein have been associated with the following diseases: high grade-glioma, breast cancer, frontal myelination, enteric nervous system, and diabetes type 1. Mutations in the gene that codes for KIT protein have been associated with the following diseases: high grade glioma, piebaldism, gastrointestinal stromal tumor (GIST), mastocytosis, liver cell membrane autoantibody (LMA), melanoma, and germ cell tumors. Mutations in the gene that codes for MLH1 protein have been associated with the following diseases: high grade glioma, hereditary nonpolyposis colorectal cancer (Lynch), and Turcot syndrome type I; see Table 6. Discussion According to the Central Brain Tumor Registry of the United States (CBTRUS), gliomas represent 25% of primary central nervous system tumors in adults and are more prevalent in men than in women. In the age range between 15 and 39 years, the oligodendrogliomas and astrocytomas predominate. For those over 40 years of age, the most frequent are glioblastomas (3). These results are consistent with our patient population, in which 21/31 patients presented with glioblastomas. The 31 patients included in this study had a mean age of 47 (SD: 14.5), and women predominated (65.4%). This result is due to the small number of patients, which is not representative of the population. Traditionally, patients with glial tumors were classified using H-E, and in some cases, the search for the mutation in the gene that codes for Isocitrate Dehydrogenase (IDH) is performed by immunohistochemistry (IHC) according to the WHO 2016 criteria (12). Based on these two analyses, we obtained the following results in the 31 patients with high-grade gliomas: 21 glioblastomas, five anaplastic astrocytomas, and five anaplastic oligoastrocytoma; see Supplementary Material 1. Currently, the diagnosis of high-grade gliomas is made not only with H-E and IHC but also includes the search for genetic mutations, epigenetic changes, and chromosomal alterations (13). Microvascular proliferation and necrosis are evaluated using H-E, atypia, mitotic activity, and increased cell density (14). Meanwhile, IHC makes it possible to verify, among other things, that tumor cells are derived from glia (15). As noted above, molecular biology studies are the cornerstones of the new 2021 WHO classification of tumors of the central nervous system and allow the classification of high-grade gliomas such as glioblastoma (GBwt-IDH), in which mutations in the TERT promoter, EGFR promoter, chromosome 7 trisomy, and monosomy chromosome 10 are frequent; Astrocytoma (Amut-IDH,^ sin-codel-1p/19q,^ G3-4), in which mutations in the ATRX, TP53 and CDKN2A/B genes can be found; and Oligodendroglioma (Omut-IDH, codel-1p/19q, G3), in which TERT, CIC, FUBP1, and NOTCH1 promoter mutations are common (7). Therefore, the group of 31 patients was reevaluated, and patients with high-grade gliomas were classified as 21 previously diagnosed as glioblastoma, of which 19 of them remained GBwt-IDH, and two changed to Amut-IDH, without codel 1p/19q, G-3,4. Of the five patients with anaplastic astrocytoma, four retained the diagnosis, and one switched to Omut-IDH, codel 1p/19q, g-3. Of the 5 patients with anaplastic oligoastrocytoma, four retained the diagnosis, and one switched to Amut-IDH, codel 1p/19q, G-3, 4, which highlights the importance of molecular methods for a correct classification of high-grade gliomas; see Supplementary Material 1. Performing an accurate diagnosis on these patients would help us provide adequate treatment, which consists of the widest surgical cytoreduction (16), followed by the Stupp Protocol (17), except for the Omut-IDH, codel -1p/19q, G3, in whom the tendency is to give only postoperative temozolomide, postponing radiotherapy (18, 19). 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337
oligoastrocytomas because, at 24 months (the end of the investigation), 5/10 patients were still alive; therefore, progression free survival (PFS) was analyzed. In the present investigation, we found that PFS at first and second relapse was higher in patients with gliomas who had the methylated promoter of the gene that codes for MGMT protein compared to those who did not; see Supplementary Material 1. These results are consistent with those reported in the literature (28). However, new biomarkers are required, which, in association with the methylation of the MGMT promoter, make it possible to predict the response of patients with high-grade glioma to the use of temozolomide. Therefore, in the present study, the detection and analysis of mutations was performed on 324 cancer- related genes in a group of 31 patients with high-grade gliomas to detect the genes that are related to the patients’ prognoses and resistance to temozolomide, finding 185 mutated genes with 370 different mutations. As a first statistical analysis, a bivariate analysis was conducted (Cox’s regression models, Kaplan-Meyer analysis, and survival curves), through which the relationship of each mutated gene with the second relapse was evaluated after the use of temozolomide as part of the Stupp protocol and metronomic dose, finding 71 genes related to second relapse; however, only the first five genes had significant values of p-value and Hazard Ratio; see Table 1. As a second statistical analysis, a multivariate analysis was performed, and Cox’s regression models were used to determine the mutated genes that were related to the second relapse. The genes with statistically significant values were PIK3C2B with a crude HR of 13.81 (95%, CI: 2.25–84.45, p = 0.004), KIT with a HR of 3. (95%, CI: 1.20–13.18, p = 0.024), ERBB3 with a HR of 3.87 (95%, CI: 1.06–14.04, p = 0.04), and MLH1 with a HR of 3.52 (95%, CI: 0.95–13.09, p = 0.06); see Table 2. Additionally, Progression Free Survival (PFS) was assessed at first and second relapse among patients with and without mutations in the PIK3C2B, ERBB3, KIT, and MLH1 genes. At the first relapse, the patients who had the mutations in the genes presented a lower PFS (8.85 months) than those who lacked the mutations (27.55 months). Also, PFS was compared for the second relapse and patients with mutations in these four genes had lower PFS (19.3 months) than those without mutations (38.17 months). Besides, it is observed that the patients who have mutations in the PIK3C2B gene had the lowest survival free progression. Therefore, presenting mutations in any of these four genes increases the probability of relapse in the patients; see Supplementary Material 1. In the study we found that PIK3C2B, ERBB3, KIT, and MLH1 genes had different types of mutations, which could affect the structure and functional domains of the proteins. Additionally, these alterations could be affecting different interactions and metabolic pathways, which could help us to hypothesize the mechanisms used by cancer cells to proliferate and acquire resistance to temozolomide treatment. After evaluating the PIK3C2B gene, a mutation, amplification, and rearrangement were found. PIK3C2B protein is involved in the biosynthetic and signaling process of phosphatidylinositol phosphatase called PIK3C2B (phosphatidylinosi-tol-4-phosphate 3-kinase C2), which is part of a family of enzymes capable of phosphorylating the hydroxyl group at the 3' position of the inositol ring of molecules, collectively called phosphatidylinositol, which convert phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3), and then to phosphorylate AKT, among others (29, 30, 31). Regarding the ERBB3 gene, two mutations were found: R164K, which affects the domain (Rcp_L) and L1177l, which does not affect any functional domain, but it affects the structure of the protein. This gene encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. This membrane-bound protein has a neuregulin binding domain but not an active kinase domain. Therefore, it can bind this ligand but not convey the signal into the cell through protein phosphorylation. However, it does form heterodimers with other EGF receptor family members which do have kinase activity. Heterodimerization leads to the activation of pathways, which lead to cell proliferation or differentiation. Amplification of this gene and/or overexpression of its protein have been reported in 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
numerous cancer types, including prostate, bladder, and breast tumors (32, 33). Regarding the KIT gene, an amplification and equivocal amplification were found. This gene encodes a receptor tyrosine kinase. It was initially identified as a homolog of the feline sarcoma viral oncogene v-kit and is often referred to as proto-oncogene c-Kit. The canonical form of this glycosylated transmembrane protein has an N-terminal extracellular region with five immunoglobulin-like domains, a transmembrane region, and an intra-cellular tyrosine kinase domain at the C-terminus. Upon activation by its cytokine ligand, stem cell factor (SCF), this protein phosphorylates multiple intracellular proteins that play a role in the proliferation, differentiation, migration, and apoptosis of many cell types and thereby plays an important role in hematopoiesis, stem cell maintenance, gametogenesis, melanogenesis, and in mast cell development, migration, and function. This protein can be a membrane-bound or soluble protein (34, 45). Regarding the MLH1 gene, the F261fs*7 mutation was found. The protein encoded by this gene can heterodimerize with mismatch repair endonuclease PMS2 to form MutL alpha, part of the DNA mismatch repair system. When MutL alpha is bound by MutS beta and some accessory proteins, the PMS2 subunit of MutL alpha introduces a single strand break near DNA mismatches, providing an entry point for exonuclease degradation. This protein is also involved in DNA damage signaling and can heterodimerize with DNA mismatch repair protein MLH3 to form MutL gamma, which is involved in meiosis (36). According to the previous biological functions, two possible mechanisms of resistance to temozolomide are postulated, in which these four genes could be involved. Regarding the first resistance mechanism, the catalytically inactive human growth factor receptor 3 (ERBB3) alone can serve as the kinase domain activating partner in a heterodimer in association with ERBB2 (Her2) (33). Its subsequently transactivated carboxyl-terminal tail domain binds to the SH2 domain of all three PI3K regulatory subunits (37). The KIT proto-oncogene transcribes the homologous receptor of the feline viral sarcoma v-kit, also called CD117 (c-Kit), which, with its binding to the physiological ligand, produces dimerization, which leads to transphosphorylation. This, in turn, reorients the domain intracellular membrane, freeing it from the autoinhibitory conformation that it acquires in an inactive state, and facilitating its catalytic function (34). Like the previous receptor, some of these phosphorylation sites in the inner membrane domain of c-Kit bind with SH2 domains, forming docking sites for signaling and activation of the pathway (PI3K, Akt, TSC1/2, mTOR1/2, and transcription factor) (38). Under physiological conditions, growth factor stimulation, in this case, neuregulin for ERBB3 (39) and stem cell factor for c-kit (40), phosphorylates enzyme-specific kinases that make up PI3K through sequential association with adapter molecules (GRB2-SOS-RAS cascade); activated PI3K phosphorylates and converts PIP2 to PIP3, located on the mid surface of the plasma membrane. Activated AKT phosphorylates and inactivates the TSC1/2 complex. This activates mTORC1 by inhibiting mTORC1 suppression mediated by the Ras homolog of GTP- binding protein enriched in the brain (RHEB) to stimulate cell proliferation and survival (29). In the RTK-PI3K-mTOR2 pathway, the latter binds to ribosomes and activates AGC subfamily kinases (glucocorticoid/serum-induced Akt kinases and PKCα), which stimulates cell migration (41). In relation to kinase enzymes, which are part of the PI3K family, and from which phosphorylate inositol phospholipids (PI)—concentrated on the cytosolic surface of membranes—are synthesized, especially in the endoplasmic reticulum, they have more than a structural function; they have important roles in cell signaling (42). Eighteen percent of the glioblastomas have a somatic mutation of the PIK3CA and PIK3R1 genes, which encode the p85 subunit and the p110 subunit of PI3K, respectively (43). Most PIK3CA and PIK3R1 mutations are frequently in the domain required for the interaction of the p85 subunit with the p110 subunit (42). Inhibition of p110 subunit activity by the p85 subunit is lost, and PI3K mutants become constitutively active, resulting in sustained AKT activity (43). PIK3C2B is a catalytic enzyme of the PI3K family, whose functions are to phosphorylate the hydroxyl group at the 3' position of the inositol ring in the plasma membrane and 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
FUNDING
This research was funded by CES University and Americas-AUNA clinic. ACKNOWLEDGMENTS The authors thank the Graduate School of the CES University for the training of doctoral students (LDOG) and Américas-AUNA clinic for al-lowing us to conduct this research. REFERENCES
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Table 2. Multivariate analysis was used to identify the mutated genes associated with the second relapse, after the use of temozolomide, according to the Stupp protocol and metronomic dose, in patients with high-grade gliomas. Gene P- value HR Raw value (CI 95%) P-value^ HR Adjusted value (CI 95%) PIK3C2B 0,004 13,81 (2,258 - 84,45) 0, 82,37 (8,36 - 811,67) KIT 0,024 3,985 (1,204 - 13,18) 0,002 10,24 (2,42 - 43,34) ERBB3 0,04 3,871 (1,066 - 14,04) 0,001 13,20 (2,77 - 62,77) MLH1 0,06 3,528 (0,950 - 13,09) 0,006 8,50 (1,83 - 39,45) 727 728 729 730 731 732 733 734 735 736 737
F1000Research Table 3. The following table shows the domains, biological processes, molecular function, and cellular components of the four proteins related to the second relapse after using temozolomide according to the Stupp protocol and a metronomic dose in patients with high-grade gliomas. 20 Protein Domains Biological processes Molecular function Cellular component PIK3C2B - R458Q mutation affects domain (PI3K_Ras- bd_dom)
- Amplification
- Rearrangement -Phosphatidylinositol-mediated signaling
- Phosphatidylinositol phosphatase biosynthetic process -Phosphatidylinositol binding -kinase activity None ERBB3 - R164K mutation affects domain (Rcp_L)
- L1177l the mutation does not affect any functional domain -Membrane receptor protein tyrosine kinase signaling pathway. -Protein phosphorylation -Protein kinase activity -Protein tyrosine kinase activity -ATP binding Membrane KIT - Amplification
- Wrong amplification -Protein phosphorylation -Transmembrane receptor protein tyrosine kinase signaling pathway -Kit signaling pathway -Fc receptor signaling pathway -ATP binding -Transmembrane receptor -Protein tyrosine kinase activity -Protein kinase activity -Cytokine binding -Protein tyrosine kinase activity None MLH1 -(F261fs*7) mutation affects domain (DNA_mismatch_repair_N and DNA_mismatch_S5_2-like) DNA mismatch repair -Mismatched DNA binding -ATP binding -ATP-dependent DNA damage Sensor activity -ATP hydrolysis activity Mismatch Repair Complex 22 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 23
