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rom four patients, representing a mutation rate of 2. 7%. In addition to PI3K activation by way of mutation, loss of PTEN represents one more mechanism by way of which the PI3K AKT pathway can grow to be activated. Hence, we also investigated the expres sion status of PTEN in GC. A total of 61 certified tumor samples from the identical cohort of Chinese GC were exam ined by IHC staining making use of an anti T0901317  PTEN antibody. As shown in Table 2 and Figure 1, the loss of PTEN protein expression was located in 23% in the tested sam ples, constant with the reported rate of 20%. Additional sequencing evaluation in the 61 samples indicated that PTEN loss overlapped with Braf mutation in a single case, but was mutually exclusive with PI3KCA and Kras mutations.
Anti tumor efficacy of AZD5363 in gastric PDGCX mouse models with AZD2858 PI3KCA mutation or PTEN loss The lack of GC patients with both PI3KCA mutations and PTEN loss as well as the higher prevalence of PTEN loss observed in GC triggered us to investigate the response of GC with PTEN loss to AZD5363. On the other hand, as a result of lack of GC cell lines with PTEN loss and wild kind PI3K, we screened 15 gastric PDGCX mouse models established from surgical samples of Chinese GC pa tients. The expression levels of PTEN protein were mea sured by IHC staining and genomic PTEN aberrations were detected by MLPA evaluation respectively. PI3KCA hotspot mutations were screened by direct sequencing. As indicated in Table four, SGC020, a PDGCX model using a PTEN exon 2 6 gene deletion and undetectable PTEN protein expression and SGC100, a PDGCX model har boring a PI3KCA H1047R activating mutation and posi tive PTEN staining, were both identified for AZD5363 anti tumor efficacy study.
As shown in Figure 2A, larger levels of basal phosphor AKT and phosphor S6 were detected by Western blot in SGC100 and SGC020 tumors in comparison with that inside the SGC001 PDGCX Lomeguatrib tu mors with PI3K and PTEN wild kind status, indicating the up regulation of AKT signal pathway in SGC100 and SGC020 models. Next we tested the response of SGC100 and SGC020 models to AZD5363. As shown in Figure 2B and 2C, AZD5363 single agent therapy resulted in 60% tumor growth inhibition in SGC100 model but had only marginal effects inside the PTEN null SGC020 model. AZD5363 therapy within this study was well tolerated and didn't lead to significant physique fat loss. These data indicate that PI3KCA mutations, but not PTEN loss, predicate the sensitivity to AZD5363 in GC.
Chemotherapy may be the current regular of care for GC. In further work, we preformed in vitro combination of AZD5363 with the normally used chemotherapy agents in GC like Taxotere, SN 38 and Oxaliplatin in a variety of GC cell lines with both PI3KCA muta tion and PTEN loss, PI3KCA mutation alone, and PI3K and PTEN wild kind status. Our data showed that the Human musculoskeletal system combination of AZD5363 with Taxotere, SN 38 and Oxaliplatin resulted in additive or slightly synergistic impact irrespective of the mutation status in PI3K gene. Previous reports have recommended a role for PTEN loss in chemotherapy resistance. Thus, we subsequent tested regardless of whether PTEN loss contributed to Taxotere resist ance, one of the main chemotherapy agents used clinic ally in GC.
As shown in Figure 2B, Taxotere GANT61 at a human equivalent dose of five mg kg weekly had no impact on tumor growth inside the T0901317  SGC020 model with PTEN loss. In contrast, combinations of AZD5363 and Taxotere resulted in significant tumor inhibition inside the PDGCX model, supporting a potential combination strategy for the therapy of GC with PTEN loss. Furthermore, the induction of caspase3 7 by combination of AZD5363 with Taxotere, the hallmark of cell apoptosis, was observed in multiple tested cell lines, suggesting the anti tumor impact of AZD5363 with Taxotere by induc tion of apoptotic cell death. Pharmacodynamic modulation of AKT signaling by AZD5363 correlates with anti tumor activity To know the mechanism of AZD5363 anti tumor efficacy and its combination with Taxotere in SGC020 and SGC100 models, tumor samples were collected two hours post last dose of AZD5363.
Tissues lysates were subjected to Western blot evaluation of PRAS40 and S6 phosphorylation, both downstream targets of AKT signal ing. As shown in Figure three, AZD5363 single agent treat ment led to up regulated GANT61 pAKT in both SGC100 and SGC020 PDGCX T0901317  models, indicating the engagement of AZD5363 with its distinct target. It is actually noteworthy that the undetectable GANT61 pAKT inside the untreated SGC100 and SGC020 tumors was due to a shorter western blot exposure because quite robust signals were detected inside the AZD5363 treated samples. Interestingly, the suppression of AKT downstream signaling monitored by pPRAS40 and pS6 was only observed inside the PI3KCA mutant PDGCX model, but not inside the PTEN null PDGCX model, correlating with AZD5363 anti tumor efficacy. Constant with our recent observa tions in cell cultures, Taxotere therapy led to a moderate enhance of pPRAS40 in SGC20 model as well as the addition of AZD5363 blocked this induction. These results further help the