Immunohistochemistry For pAKT Research Articles

Exosomes isolated from IMMUNEPOTENT CRP, a hemoderivative, to accelerate diabetic wound healing.

The increasing risk of amputation due to diabetic foot ulcer calls for new therapeutic options; for that, we determined the role of IMMUNEPOTENT CRP (ICRP) and its parts in the wound healing process of superficial wounds in diabetic BALB/c mice. A potency test was performed to confirm the batch of ICRP, and then its parts were separated into pellets, supernatants, and exosomes, and another group of exosomes loaded with insulin was added. Viability and scratch healing were assessed in NIH-3T3, HUVEC, and HACAT cell lines. Diabetes was induced with streptozotocin, and wounds were made by dissecting the back skin. Treatments were topically applied, and closure was monitored; inflammatory cytokines in sera were also evaluated by flow cytometry, and histological analysis was performed by Masson's staining and immunohistochemistry for p-AKT, p-FOXO, p-P21, and p-TSC2. ICRP pellets and exosomes increased cellular viability, and exosomes and exosome-insulin accelerated scratch healing in vitro. Exosome-insulin releases insulin constantly over time in vitro. In vivo, treatments accelerated wound closure, and better performance was observed in pellet, exosome, and exosome-insulin treatments. Best collagen expression was induced by ICRP. P-AKT and p-FOXO were overexpressed in healing tissues. Inflammatory cytokines were downregulated by all treatments. In conclusion, IMMUNEPOTENT CRP components, especially exosomes, and the process of encapsulation of exosome-insulin accelerate diabetic wound healing and enhance cellular proliferation, collagen production, and inflammation modulation through the phosphorylation of components of the AKT pathway.

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro.

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses (fresh control) or cultured in α-MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL-positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α-MEM+ and 10 μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.

Abstract OT3-06-06: A phase I/II study of preoperative letrozole, everolimus, and TRC105 in women with newly diagnosed local or locally advanced potentially resectable hormone-Receptor positive and Her2 negative breast cancer

Abstract Hypothesis: Pharmacologic inhibition of angiogenesis and mTOR pathway blockade will enhance the efficacy of aromatase inhibitors in the neoadjuvant setting in women with hormone receptor-positive and Her2-negative (HR+/Her2-) breast cancer. Background: In non-metastatic HR+/Her2- breast cancer, downstaging to stage I or 0 in the neoadjuvant setting has been associated with favorable prognosis. With chemotherapy yielding similar benefits with endocrine therapy, aromatase inhibitors have become the agents of choice in postmenopausal women. The elucidation of pathways that are operational in luminal breast cancer and compromise the efficacy of hormonal therapy along with preclinical and clinical evidence of successful combinations of endocrine therapy with targeted agents set the stage for more rational, effective, and equally well tolerated regimens. Proangiogenic signaling has been strongly associated with accelerated hormone-independent growth and resistance to endocrine therapies. Breast cancer cells with upregulated PI3K/AKT/mTOR pathway are resistant to hormonal therapy and this resistance has been restored by everolimus. Inhibiting mTOR, a downstream signaling node where multiple pathways converge has the potential to abrogate primary and escape signaling cascades that mediate resistance to endocrine therapies. Study Design: Phase I dose escalation: follows a 3+3 design. During the phase I, the Maximum Tolerated Dose (MTD) and Recommended Phase 2 Dose (RP2D) will be determined. Letrozole will be kept the same for all cohorts (2.5 mg PO qd). Everolimus will be escalated from 5 mg (cohort 1) to 10 mg PO qd (cohort 2). TRC105 will be 15 mg/kg q2 weeks (cohorts 1 and 2). Premenopausal women at the time of enrollment will receive goserelin 3.6 mg SC q4 weeks to achieve ovarian suppression. Duration of treatment is 24 weeks. Phase II dose expansion: will be initiated once the MTD and RP2D have been determined. Phase II follows a Gehan's two-stage design: 10 patients will enroll first, and if no patient has achieved downstaging, the study will close since it is unlikely (p=0.1) that 0/10 responses would occur if true response rate were &amp;gt;20%. If at least one patient has downstaging, 10 additional patients will enroll (total, 20). Primary Objectives Phase I: Determine the tolerability and feasibility of letrozole, everolimus, and TRC105 in women with newly diagnosed stage 2 and 3 HR+/Her2- breast cancer. Phase II: in the same patient population determine the efficacy, pharmacokinetic, and pharmacodynamic parameters of the combination. Correlative Studies: Immunohistochemistry for pAKT and PTEN, CD105, CD31/CD34, NG2, MCAM/CD146 on diagnostic tissue, research biopsy, and final surgical specimen. Surgical specimens that meet criteria for next-generation sequencing will undergo ribosome profiling Key Eligibility Criteria: Recent diagnosis of HR+Her2- breast cancer Stage 2 and 3 breast cancer Any histological grade No prior treatment specific for breast cancer Pre- and perimenopausal women are eligible if ovarian suppression is achieved with goserelin. Current Status: Cohort 1 of the Phase 1 complete. Cohort 2 of Phase 1 actively enrolling. Citation Format: Vaklavas C, Lamaster KV, Stringer EM, Falkson CI, Li Y, Forero A. A phase I/II study of preoperative letrozole, everolimus, and TRC105 in women with newly diagnosed local or locally advanced potentially resectable hormone-Receptor positive and Her2 negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-06-06.

MTOR inhibitors response and mTOR pathway in pancreatic neuroendocrine tumors.

Medical therapy of pancreatic neuroendocrine tumors (P-NET) may take advantage of Everolimus treatment. However, the extent of therapeutic response cannot be predicted. This study was aimed to identify the possible predictive markers of response to Everolimus in P-NET. We found that Everolimus reduced the cell viability and induced apoptosis in primary cultures of 6 P-NET (P-NET-R), where the proliferative and antiapoptotic effects of IGF1 were blocked by Everolimus. On the contrary, 14 P-NET primary cultures (P-NET-NR) were resistant to Everolimus and IGF1, suggesting an involvement of PI3K/AKT/mTOR pathway in the mechanism of resistance. The response to Everolimus in vitro was associated with an active AKT/mTOR pathway and seemed to be associated with a greater clinical aggressiveness. In addition, a patient sensitive to Everolimus in vitro was sensitive to this drug in vivo also and showed a positive p-AKT immunohistochemistry (IHC) at tissue level. Similarly, a patient resistant to Everolimus treatment after surgery was not sensitive to the drug in vitro and had a negative p-AKT IHC staining. Therefore, present data confirm that P-NET primary cultures may be considered a model for testing medical treatment efficacy and that IHC characterization of p-AKT might help in identifying human P-NET who can benefit from Everolimus treatment. These data encourage conducting a prospective multicenter study involving different groups of P-NET patients treated with Everolimus.

Activation of Akt at T308 and S473 in alcohol, tobacco and HPV-induced HNSCC: is there evidence to support a prognostic or diagnostic role?

BackgroundTobacco, alcohol and HPV infection are associated with increased risk of HNSCC. However, little is known about the underlying signaling events influencing risk. We aimed to investigate the relationship between these risk factors and Akt phosphorylation, to determine prognostic value.MethodVEGF-positive HNSCC biopsies, with known HPV status, were analyzed by immunohistochemistry (IHC) for Akt, phosphorylated at residues S473 and T308. Comparisons between the tissues were carried out using a Mann–Whitney U test. Associations between the variables and continuous immunohistochemical parameters were evaluated with general linear models. Patient characteristics and pAkt IHC score were analyzed for possible association with overall survival by Cox proportional hazard models.ResultsImmunohistochemistry revealed that cancer patients had significantly higher levels of pAkt T308 than S473 (P < 0.001). Smoking and alcohol were found to be independent risk factors for Akt phosphorylation at T308 (P = 0.022 and 0.027, respectively). Patients with tumors positive for HPV or pAkt S473 had a poorer prognosis (P = 0.005, and 0.004, respectively). Patients who were heavy drinkers were 49 times more likely to die than non-drinkers (P = 0.003). Patients with low pAkt T308 were more likely to be HPV positive (P = 0.028). Non-drinkers were also found to have lower levels of pAkt T308 and were more likely to have tumors positive for HPV than heavy drinkers (P = 0.044 and 0.007, respectively).ConclusionThis study suggests different mechanisms of carcinogenesis are initiated by smoking, alcohol and HPV. Our data propose higher phosphorylation of Akt at T308 as a reliable biomarker for smoking and alcohol induced HNSCC progression and higher phosphorylation of Akt at S473 as a prognostic factor for HNSCC.

High rate of FGFR1 amplifications in brain metastases of squamous and non-squamous lung cancer

ObjectivesFGFR1 amplifications are common in squamous cell carcinoma and rare in adenocarcinoma of the lung, but have not been investigated in brain metastases of non-small cell lung cancer (NSCLC). Materials and methodsWe performed fluorescent in situ hybridization (FISH) for FGFR1 and immunohistochemistry for pAKT, PI3K, HIF1a and Ki67 in 175 NSCLC brain metastases and 11 matched primary tumors. ALK gene rearrangement status was available from a previous study. We performed statistical correlations of clinical, histopathological and molecular data. ResultsFGFR1 amplifications were found in a total of 30/175 (17%) brain metastases: 4/21 (19%) squamous cell carcinomas, 20/130 (15.3%) adenocarcinomas, 2/12 (16.6%) adenosquamous carcinomas, 4/9 (44.4%) large cell carcinomas and 0/3 neuroendocrine large cell carcinoma. FGFR1 gene status was identical between primary tumors and brain metastases in 9/11 evaluable cases. In 2/11 cases (1 adenosquamous and 1 large cell carcinoma), FGFR1 amplifications were present only in the brain metastasis and not in the primary tumor. Furthermore, we found a significant positive correlation of ALK and FGFR1 gene amplification status in brain metastases (p<0.001, Chi square test). Patients with high-level FGFR1 amplifications had significantly higher number of visceral metastases (p<0.001, Chi square test). ConclusionOur findings argue for an enrichment of FGFR1 amplifications in brain metastases of adenocarcinomas (where they were 5-fold more frequent than reported for primary tumors) and possibly also of other non-squamous carcinomas, but not in squamous cell carcinomas of the lung. These results may be relevant for targeted therapy and prophylaxis of NSCLC brain metastases.

Immunohistochemical Analysis of the Activation Status of the Akt/mTOR/pS6 Signaling Pathway in Oral Lichen Planus

Introduction. Aberrations of the Akt/mTOR/pS6 pathway have been linked to various types of human cancer, including oral squamous cell carcinoma (OSCC). The purpose of this study was to evaluate the activation status of Akt, mTOR, and pS6 in oral lichen planus (OLP) in comparison with oral premalignant and malignant lesions and normal oral mucosa (NM). Materials and Methods. Immunohistochemistry for p-Akt, p-mTOR, and phospho-pS6 was performed in 40 OLP, 20 oral leukoplakias (OL), 10 OSCC, and 10 control samples of NM. Results. Nuclear p-Akt expression was detected in the vast majority of cases in all categories, being significantly higher in OL. Cytoplasmic p-Akt and p-mTOR staining was present only in a minority of OLP cases, being significantly lower compared to OL and OSCC. Phospho-pS6 showed cytoplasmic positivity in most OLP cases, which however was significantly lower compared to OL and OSCC. Conclusions. Overall, cytoplasmic p-Akt, p-mTOR, and phospho-pS6 levels appear to be significantly lower in OLP compared to OL and OSCC. However, the expression of these molecules in a subset of OLP cases suggests that activation of Akt/mTOR/pS6 may occur in the context of OLP, possibly contributing to the premalignant potential of individual cases.

PAKT expression in paraffin-embedded xenograft tumors after fixation delays and human breast cancer by optimized immunohistochemistry.

10603 Background: With progress in targeting PI3K/AKT pathway in the treatment of a wide range of cancers, it is getting more important than ever for optimization of detection method of AKT kinase, a central effector of the pathway, in archived tissues. Recently, we found that pAKT-S473 (pAKT) significantly predicts paclitaxel benefit in breast cancer (JCO 28: 2974, 2010), in which a good analytical and clinical performance of quantitative pAKT immunohistochemistry (IHC) was demonstrated. The current study evaluates pAKT stability in human breast cancer xenograft tumors after delays in fixation, and assesses a detection window following fixation delays with an established cutoff [staining index (SI) &gt; 2]. Methods: Xenograft tumors with high (MDA-MB-468) and intermediate levels (MDA-MB-231) of pAKT were fixed or snapped frozen in 10% neutral-buffered formalin or liquid nitrogen after delays post-excision. pAKT staining was performed in xenograft tumors and 96 cases of human surgical breast tumors by our optimized and traditional IHCs, and Western blot. Results: The mean SIs were 133, 121, 112, 94, 84, and 66 using optimized, and in contrast were 51, 44, 29, 14, 12, and 6.4 on traditional method at 0, 15, 30, 60, 120 and 180 min (2.6-fold by comparing the optimized with the traditional at baseline) in MDA-MB-468 xenograft tumors. pAKT level was ½ by the optimized, similar to the level detected by Western blot, relative to 1/8 of the baseline by the traditional (10.6-fold) at 180 min. The logarithmic decline rate by the optimal was 3.1 times (95% CI, 2.4 - 3.7) less than that of the traditional (normal approximation; 2-sided P &lt; 0.0001). pAKT expression was observed in 38.5% (37/96 ) of surgical breast tumors, comparable to 38% (606/1581) in a large cohort derived from the NSABP B-28 trial. There was little loss of pAKT in MDA-MB-231 xenograft tumors up to 180 min by IHC and Western blot. Conclusions: The optimized pAKT IHC significantly increases the sensitivity of detection with a large window for positivity. It is suitable for use in archived human specimens although it warrants further standardization and validation among research laboratories and perhaps diagnostic laboratories in the future.

Highlights of This Issue

[18F]FLT (3′-Fluoro-3′ deoxythymidine)–positron emission tomographic imaging ([18F]FLT–PET) has been used to measure in vivo tumor cell proliferation; however, its use in clinical tumor staging is limited due to the lower overall uptake of FLT. Zhang and colleagues evaluated the factors other than tumor cell proliferation that could affect the outcome of imaging to understand the translational aspects of the [18F]FLT–PET imaging modality. The preclinical use of [18F]FLT–PET imaging for tracking therapeutic response was also assessed. The results of this study strongly agreed with the clinical outcome. This work highlights the preclinical understanding of [18F]FLT–PET imaging and provides insights into the technologic applications in the oncology arena.Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). Miyazaki and colleagues addressed the hypothesis that a G-quadruplex ligand, telomestatin (TMS), eradicates GSCs. TMS treatment induced apoptosis of GSCs in vitro and in vivo through both telomeric and nontelomeric DNA damage. A proto-oncogene, c-Myb, was identified as a novel target of TMS by the cDNA microarray and pharmacodynamic analysis. c-Myb knockdown significantly reduced the growth of GSCs both in vitro and in vivo, and c-Myb expression in surgical specimens of GBM patients was statistically significantly elevated. These data suggest a novel GSC-directed therapeutic strategy for GBM through telomere disruption and c-Myb inhibition.The prostate cancer field is critically lacking a metastasis xenograft model that respects the entire physiologic metastatic cascade and provides evidence for its clinical relevance. Lange and colleagues established subcutaneous xenograft models using 4 prostate cancer cell lines and quantified tumor development and spontaneous metastasis formation. Metastatic xenografts revealed an increased activity of Mgat5b, a β(1,6)- branching glycosyltransferase previously found in neurons and testes, as detected by Phaseolus vulgaris leukoagglutinin (PHA-L) binding. According to a clinical part of the study (n = 2,085), PHA-L intensity was correlated with serum prostate-specific antigen (PSA) levels, and a cytoplasmic localization of PHA-L binding adversely affected PSA recurrence-free survival.Few studies have explored the connection between tumor imaging results and gene expression. To identify signaling pathways associated with treatment-related changes in tumor 2[18F]fluoro-2-deoxy-D-glucose–positron emission tomographic (FDG–PET) imaging, Schwarz and colleagues performed gene expression profiling using 62 pretreatment cervical cancer biopsies from patients treated with definitive chemoradiation. All patients underwent pre- and post-treatment FDG–PET. Alterations in expression of genes from the phosphoinositide 3-kinase (PI3K)/Akt pathway were associated with abnormal FDG uptake on the posttreatment PET. These results were validated using immunohistochemistry for p-Akt and comparing results with patient survival outcome data after treatment (n = 174). These results suggest that PI3K/Akt inhibition may improve response to chemoradiation.

PI3K signaling pathway is activated by PIK3CA mRNA overexpression and copy gain in prostate tumors, but PIK3CA, BRAF, KRAS and AKT1 mutations are infrequent events

The phosphatidylinositol 3-kinase (PI3K)–AKT and RAS–MAPK pathways are deregulated in a wide range of human cancers by gain or loss of function in several of their components. Our purpose has been to identify genetic alterations in members of these pathways in prostate cancer. A total of 102 prostate tumors, 79 from prostate cancer alone (group G1) and 23 from bladder and prostate cancer patients (G2), are the subject of this study. In 20 of these 23, the bladder tumors were also analyzed. PIK3CA, KRAS, BRAF and AKT1 mutations were analyzed by direct sequencing, and BRAF also by pyrosequencing. PIK3CA quantitative mRNA expression and fluorescence in situ hybridization (FISH) gains were tested in 25 and 32 prostate tumors from both groups (G1 and G2), respectively. Immunohistochemistry for pAKT was performed in 55 prostate tumors. Of 25 prostate tumors, 10 (40%) had PIK3CA mRNA overexpression that was statistically associated with Gleason score ≥7 (P=0.018). PIK3CA copy gain was detected in 9 of 32 (28%) prostate tumors. Of 20 bladder tumors, 3 (15%) displayed mutations in PIK3CA, KRAS and AKT1, the corresponding prostate tumors being wt. We also detected a previously not reported PIK3CA polymorphism (IVS9+91) in two prostate tumors. In all, 56% of prostate tumors overexpressed pAKT. There is a statistical association (P<0.0001) of strong pAKT immunostaining with high Gleason score, and with PIK3CA alterations (mRNA overexpression and/or FISH gains). PIK3CA gene is deregulated by mRNA overexpression and DNA gain in ∼40 and 28% of prostate tumors, respectively. High-grade prostate tumors are associated with PIK3CA mRNA overexpression, but not with FISH status. PIK3CA, BRAF, KRAS and AKT1 mutations are very infrequent events in prostate tumors. However, PI3K signaling pathway is activated by PIK3CA FISH gain and/or mRNA overexpression, leading to an increased pAKT protein expression.

Extracellular signal-regulated kinase and phosphatidylinositol-3-kinase/AKT signalling pathways in the human carotid body and peripheral ganglia

Extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K)/AKT signalling pathways are involved in various cell functions, but their developmental regulation in the carotid body and peripheral ganglia has not yet been fully investigated. ERK and AKT immunolocalisation and activation were studied by anti-ERK, -pERK, -AKT and -pAKT immunohistochemistry in carotid bodies and peripheral (sympathetic and sensory) ganglia, sampled at autopsy from 4 foetuses (mean gestational age 177 days), 8 infants (mean age 10 months), 8 young adults (mean age 38 years) and 6 aged adults (mean age 72.4 years). ERK and AKT immunopositivity and activation were demonstrated in both glomic type I cells and peripheral ganglionic cells and are ascribed to local action by neuromodulators or neurotrophic factors. Mean percentages of ERK- and pERK-immunopositive glomic type I cells were lower in foetuses than in infants and young adults, and those of AKT-immunopositive glomic type I cells were lower in foetuses than in young and old adults, suggesting incomplete maturation of these two signalling pathways in foetal life. Both pERK and pAKT immunoreactions were detected only in post-natal sympathetic and sensory ganglia, demonstrating that, also in peripheral ganglia, these pathways are not yet fully operative during the foetal stage.

Dietary sphingolipids suppress a subset of preneoplastic rat liver lesions exhibiting high PTEN, low phospho-Akt and high levels of ceramide species

Rat liver glutathione- S-transferase Pi-(GST-P)-positive enzyme-altered foci (EAF) are preneoplastic lesions that develop in response to carcinogenic stress. They are often used as endpoints in e.g. chemopreventive studies. In this study we characterize a pAkt-negative/ceramide-positive (pAkt−/cer+) EAF phenotype, as defined by immunohistochemistry for pAkt and ceramide species, in diethylnitrosamine(DEN)-, phenobarbital- or aflatoxinB1-treated rats. There was a close to 100% overlap for the pAkt and the ceramide marker. Furthermore, serial sections stained for PTEN indicated a close correlation between PTEN-positive and pAkt-negative lesions in DEN-treated rats. Experiments with DEN-treated rats given sphingomyelin in the diet suggested that sphingomyelin selectively targeted these lesions. In in vitro experiments sphingosine rapidly decreased pAkt levels in hepatocytes, and in experiments with hepatocytes from DEN-treated rats sphingosine selectively killed EAF cells. Furthermore, pretreatment with antisense Akt oligonucleotides in vitro sensitized non-EAF hepatocytes, so that EAF and non-EAF cells became equally sensitive to sphingosine. It is concluded that rat liver, in response to carcinogenic stress, develops a distinct EAF phenotype exhibiting low pAkt levels and concomitant alterations in sphingolipid metabolism. Our data also suggest that pAkt−/cer+ EAF are selectively targeted by sphingolipids in the diet and that lesions with this phenotype should be of particular interest for future studies on chemopreventive effects that may affect sphingolipid metabolism.