Morphoproteomic Evidence of Constitutively Activated and Overexpressed mTOR Pathway in Cervical Squamous Carcinoma and High Grade Squamous Intraepithelial Lesions

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.     

Morphoproteomic and molecular concomitants of an overexpressed and activated mTOR pathway in renal cell carcinomas

CCI-779 (temsirolimus), an ester of rapamycin and an inhibitor of the mammalian target of rapamycin (mTOR), is currently in phase II trials for treatment of patients with solid cancers. The mTOR functions as a checkpoint for cell proliferation, with upstream Akt and downstream p70S6K serving as its most important mediators. The aim of this study was to evaluate the expression and activation of the Akt-mTOR-p70S6K pathway in renal cell carcinoma (RCC), seeking to strengthen the rationale for targeted therapy of RCC using rapamycin or a rapamycin analogue. Tissue microarray sections containing 128 primary RCCs, 22 metastatic RCCs, and 24 non-neoplastic (normal) kidneys (NK) were immunostained with monoclonal antibodies to phosphorylated (p)-Akt (Ser473), p-mTOR (Ser2448), and p-p70S6K (Thr389). Western blotting was performed on 3 cases of clear cell RCC (CRCC) and the corresponding non-neoplastic (normal) renal tissues using the same antibodies. The immunostain scoring system included: (a) location; (b) distribution; and (c) intensity. The normal kidneys provided baseline scores for comparison. Expression of p-Akt, p-mTOR, and p-p70S6K was seen in 100% (n = 24) of NKs and nearly 100% (n = 150) of both primary and metastatic RCCs. The p-p70S6K was located in the nucleus in both NKs and RCCs. The p-Akt was observed in the nucleus and cytoplasm of NKs and in the nucleus and cytoplasm/ membrane (plasmalemma) of RCCs. The p-mTOR was identified in the membrane of NKs and the membrane/nucleus of RCCs. The levels of expression of p-p70S6K, p-mTOR, and p-Akt were significantly higher in RCC than in NK in the overall pattern (intensity and distribution, p <0.05). Western blotting also showed higher expression of p-p70S6K, p-mTOR, and p-Akt in RCCs compared to the corresponding normal kidney tissues (p <0.05). These findings indicate that correlative over-expression and activation of p-Akt, p-mTOR, and p-p70S6K are commonly observed in RCCs. After considering these findings in the context of other established protein circuitries and pathways in RCC, we propose therapeutic approaches that incorporate rapamycin-like agents and other small molecule inhibitors in a combinatorial fashion in future clinical trials for RCC.     

Morphoproteomic confirmation of a constitutively activated mTOR pathway in high grade prostatic intraepithelial neoplasia and prostate cancer

Preclinical studies have implicated the mammalian target of rapamycin (mTOR) pathway in the cell cycle progression and growth of prostate cancer cells. Downstream signaling from PI3'-K/Akt leads to phosphorylation (p) of mTOR at serine 2448 and to activation of its substrate, p70S6Kinase (p70S6K), phosphorylated on threonine 389. This promotes translation and cell cycle progression. Morphoproteomic analysis, that combines both the application of phosphospecific probes directed against putative sites of activation on protein analytes and cellular compartmentalization [1] was carried out on tissue microarray (TMA) slides from 64 cases of primary, previously untreated adenocarcinomas of the prostate. Gleason scores ranged from 6 to 10. High grade prostatic intraepithelial neoplasia (HGPIN), which accompanied the invasive cancer in 20 cases, and 15 non-neoplastic controls from benign prostatic hypertrophy specimens in a separate TMA were also included. Ninety-three percent (93%) of tumors exhibited moderate to strong cytoplasmic/plasmalemmal expression of p-mTOR and eighty-five percent (85%) showed similar staining intensity for p-p70S6K. HGPIN demonstrated comparable and occasionally, stronger expression levels for these protein analytes. Quantitative digital imaging revealed an overall increase in the mean expression levels in HGPIN, reaching statistical significance for p-mTOR (Ser 2448) at p<0.05. Morphoproteomic analysis confirms the constitutive activation of the mTOR pathway in prostate cancer and HGPIN, with relative overexpression of p-mTOR in HGPIN. These findings coincide with preclinical studies in supporting a role for the mTOR pathway in the biology and development of prostate cancer through its putative precursor lesion, HGPIN and in suggesting a potential therapeutic target.     

Morphoproteomic profile of mTOR, Ras/Raf kinase/ERK, and NF-kappaB pathways in human gastric adenocarcinoma

Preclinical studies using human gastric adenocarcinoma (GAC) cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, can inhibit tumor growth and that the extracellular signal-regulated kinase (ERK) of the Ras/Raf kinase/ERK pathway is related to chemoresistance and apoptosis. We examined the state of activation of components of mTOR, Ras/Raf kinase/ERK, and nuclear factor (NF)-kappaB signal transduction pathways, as well as cell cycle protein analyte correlates in GAC cases. Formalin-fixed paraffin-embedded tissue microarray blocks containing samples from 210 cases of GAC were examined. Immunohistochemistry was utilized to detect the following antigens: S100P, upstream stimulator of ERK, and NF-kappaB pathways; phosphorylated (p)-mTOR (Ser 2448), p-ERK-1/2 (Thr 202/Tyr 204), and one of their common down-stream effectors, p-p70S6K(Thr 389); p-NF-kappaBp65(Ser 536); and cell cycle associated proteins, Ki-67, and S phase kinase-associated protein (Skp)2. Immunoreactivity (0 to 4+) of protein expression and compartmentalization were assessed by bright-field microscopy. The majority of cases showed positive (1+ to 4+) cytoplasmic/plasmalemmal p-mTOR (88%), and moderate-strong (2+ to 4+) nuclear p-p70S6K (93%) and nuclear S100P (81%) expression. A subset of cases exhibited moderate-strong nuclear p-ERK-1/2 (15%) and p-NF-kappaBp65 (36%) expression. The majority of cases showed concomitant moderate-strong (2+ to 4+) nuclear Ki-67 (71%) and Skp2 (68%). Nuclear expression levels of p-ERK-1/2 and p-NF-kappaBp65, of p-p70S6K and p-NF-kappaB, and of Ki-67 and Skp2, respectively, showed significant linear correlations in GAC (p <0.001). Additionally, there were statistically significant differences in the mean expression levels of p-ERK-1/2 and p-NF-kappaBp65 in diffuse vs intestinal types of GAC, with higher levels of both in the diffuse type ( p = 0.001 and p <0.0001, respectively). In summary, morphoproteomic analysis reveals constitutive activation of mTOR and to some extent, Ras/Raf kinase and NF-kappaB pathways in GAC, as evidenced by increased cytoplasmic p-mTOR, nuclear translocation of p-p70S6K and p-ERK-1/2 phosphorylated at putative sites of activation (Ser 2448, Thr 389, and Thr 202/Tyr 204, respectively), as well as correlative expression of cell cycle analytes, Ki-67, and Skp2. These results suggest that a prospective study is warranted to evaluate the use of morphoproteomic profiling of individual patients with GAC in order to design combinatorial treatment strategies that target the mTOR, Ras/Raf kinase/ERK, and/or NF-kappaB pathways.     

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma

The mammalian target of rapamycin (mTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.     

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.     

Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.     

Differential expression of mTOR signaling pathway proteins in lichen planopilaris and frontal fibrosing alopecia

Dysregulation of the mammalian target of rapamycin (mTOR) signaling pathway has a variety of effects on the immune system and stem cell proliferation. Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are inflammatory scalp conditions resulting in permanent alopecia, which are thought to be related to stem cell damage. Here we investigate the expression of mTOR signaling pathway proteins in human hair follicles of LPP and FFA patients. The expression of mTOR pathway proteins in biopsy specimens from lesional and non-lesional scalp areas of eight LPP and five FFA patients were compared to control scalp biopsies from patients undergoing surgical excisions of sebaceous cysts. We performed immunohistochemical evaluation using a panel of antibodies including mTOR, phospho-mTOR (Ser2448), phospho-p70S6K (Thr389), phospho-4EBP1 (Thr37146), and phospho-tuberin (T1462), as well as Western blot analysis for phospho-p70S6K (Thr389) expression. All evaluated mTOR pathway proteins were similarly expressed in the control and patient non-lesional scalps. While mTOR expression did not show significant alterations between the groups, p-mTOR, p-p70S6K, p-4EBP1, and p-tuberin expressions decreased in the interfollicular epidermis in the lesional scalps of patients. p-p70S6K and p-4EBP1 expression decreased in the outer root sheath (ORS) and inner root sheath (IRS) of the bulge of hair follicles in the lesional scalps of patients. p-mTOR and p-p70S6K expression increased in the lower follicle ORS and bulb of the hair follicles, and p-4EBP1 expression decreased in the bulb of the hair follicles in the lesional scalps of patients. Phospho-tuberin expression increased in the IRS of the bulge and lower follicle ORS of the hair follicles in the lesional scalps of patients, whereas its expression decreased in the bulb. Our results indicate that the mTOR signaling pathway proteins are localized throughout normal hair follicles and that expression of mTOR signaling pathway proteins is altered in the hair follicles of LPP and FFA patients. Further research is required to understand the mechanism by which mTOR operates in the pathogenesis of these diseases.     

Constitutive activation with overexpression of the mTORC2-phospholipase D1 pathway in uterine leiomyosarcoma and STUMP: morphoproteomic analysis with therapeutic implications

The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for therapy and drug development. Biologically, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR complex 1 (mTORC1) which is predominantly cytoplasmic in subcellular localization and mTOR complex 2 (mTORC2) which is both cytoplasmic and nuclear. mTORC1 is sensitive to the selective inhibitor rapamycin. By contrast, mTORC2 is relatively resistant to rapamycin. Moreover, its putative downstream effector, Akt phosphorylated on serine 473 represents a signal transduction pathway for tumor survival. Phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as an activator of mTOR signaling, including the direct phosphorylative activation of p70S6K atthreonine 389. The latter promotes cell cycle progression. In this study, we investigated the activation status and subcellular localization of mTOR and the relative expression of PLD1, as well as their downstream effectors in a spectrum of uterine smooth muscle tumors using normal myometria as controls. The results show significant activation with overexpression of phosphorylated mTORC2 complex in uterine leiomyosarcoma (ULMS) and smooth muscle tumors of uncertain malignant potential (STUMP) as evidenced by nuclear localization of p-mTOR (Ser 2448) in ULMS>STUMP>uterine leiomyoma and normal myometria (p<0.05) and with overexpression of PLD1(p<0.05). Cor-relatively, there are overexpressions of nuclear p-Akt (Ser 473) and nuclear p-p70S6K (Thr 389) in ULMS and STUMP (p<0.05). The activation with overexpression of components of the mTORC2-PLD1 pathway in ULMS and to a lesser degree in STUMP provides insight into their tumorigenic mechanisms. Thus the development of therapies designed to target mTORC2 and PLD1 activity may be beneficial in treating ULMS.     

Overexpression of phosphorylated nuclear factor-kappa B in tonsillar squamous cell carcinoma and high-grade dysplasia is associated with poor prognosis.

Intracellular signals along the epidermal growth factor receptor (EGFR)-Akt-nuclear factor-kappa B (NF-kappaB) pathway have been associated with carcinogenesis in various malignant neoplasms. This investigation was to evaluate the expression of EGFR, phosphorylated(p)-Akt and p-NF-kappaB and correlate them with clinical outcomes in patients with squamous cell carcinoma of the tonsil. A total of 45 patients with squamous cell carcinoma of the tonsil were studied by immunohistochemistry to evaluate the expression levels of EGFR, p-Akt and p-NF-kappaB. Results for squamous cell carcinoma of the tonsil were compared with those for associated high-grade dysplasia and adjacent normal appearing epithelium, when present. In addition, tonsillar epithelium from non-neoplastic specimens of age-matched patients also was stained for the same markers. High-grade dysplasia and squamous cell carcinoma of the tonsil demonstrated a similar pattern of expression, which differed from the pattern seen in the adjacent normal epithelium and tonsillar epithelium from normal controls (an overexpression for each of these three protein analytes in high-grade dysplasia and squamous cell carcinoma of the tonsil as demonstrated by immunohistochemistry). When markers from squamous cell carcinoma of the tonsil were correlated with survival status, only increasing levels of p-NF-kappaB immunoreactivity (a relative overexpression) were statistically significant predictors of poor survival. No markers in squamous cell carcinoma of the tonsil were significantly related to rate of recurrence. When analyzing marker scores from tissue with high-grade dysplasia, relative overexpressions of both p-Akt and p-NF-kappaB were significantly related to poor survival. Additionally, increasing levels of p-NF-kappaB immunopositivity from tissue with high-grade dysplasia were also significantly related to rate of recurrence. In summary, p-NF-kappaB, overexpressed in high-grade dysplasia and squamous cell carcinoma of the tonsil, is associated with worse prognosis in terms of high recurrence and poor survival, respectively. This significant finding in patients with squamous cell carcinoma of the tonsil, in combination with previous animal and in vitro studies, suggests that p-NF-kappaB represents a potential therapeutic target in head and neck squamous cell carcinoma.     

Phosphorylated mTOR expression correlates with podoplanin expression and high tumor grade in esophageal squamous cell carcinoma

Mechanistic (or mammalian) target of rapamycin (mTOR) plays important roles in cell growth and proliferation. In esophageal squamous cell carcinoma (SCC), high expression of phosphorylated (activated) mTOR (p-mTOR) has been reported as an adverse prognostic factor in some but not all studies. The signals of mTOR pathway and mitogen-activated protein kinase (MAPK) pathway converge on 4E-binding protein 1 (4EBP1), which drives the downstream proliferative signals. We previously found that high expression of phosphorylated 4EBP1 (p-4EBP1) is an adverse prognostic factor in esophageal SCC. Podoplanin is a type-1 transmembrane glycoprotein expressed in various normal human tissues, including lymphatic endothelium. Our previous study showed that high podoplanin expression correlates with clinical nodal metastasis, which is associated with short survival in esophageal SCC. In current study, we investigated p-mTOR expression by immunohistochemistry in 75 cases of surgically resected esophageal SCC. The result was correlated with p-4EBP1 expression, podoplanin expression, clinicopathologic features and patient survival. We found that high p-mTOR expression was significantly associated with high podoplanin expression (P = 0.0030) and high tumor grade (P = 0.0014). No correlation with p-4EBP1 expression, patient survival or other clinicopathologic features was found. Recently, podoplanin expression in astrocytic brain tumors was found to be regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT/activator protein-1 (AP-1) pathway. Similarly, mTOR is activated by a PI3K/AKT/mTOR pathway. The association of p-mTOR and podoplanin expression in our study could be due to a common upstream pathway. Since both mTOR and podoplanin are potential therapeutic targets, the possible benefit of combined targeted therapy warrants further investigation.     

Differential expression of fatty acid synthase (FAS) and ErbB2 in nonmalignant and malignant oral keratinocytes

The aim of this study was to investigate fatty acid synthase (FAS) and ErbB2 expression in nonmalignant oral epithelium and oral or head and neck squamous cell carcinomas (OSCC/HNSCC). Morphologically normal, hyperkeratotic, and dysplastic oral epithelium as well as well-differentiated and poorly differentiated OSCC were immunohistochemically evaluated for FAS, ErbB2, and Ki-67. These proteins were also analyzed in a tissue microarray with 55 HNSCC. SCC-9 cells were used to study FAS and ErbB2 during differentiation. FAS expression was higher in hyperkeratosis, dysplasias, and OSCC than in normal epithelium. Well-differentiated OSCC/HNSCC were more positive for FAS than the poorly differentiated tumors. ErbB2 was observed at the surface of nonmalignant and well-differentiated OSCC/HNSCC keratinocytes and in the cytoplasm of poorly differentiated cells. Ki-67 index was progressively higher from normal oral epithelium to OSCC, inversely correlated with cell surface ErbB2, and positively correlated with intracytoplasmic ErbB2. Finally, SCC-9 cell cultures were enriched in membrane ErbB2-positive cells after differentiation by anchorage deprivation. In conclusion, FAS is overexpressed in OSCC/HNSCC and hyperkeratotic oral epithelium and ErbB2 is found at the cell surface of differentiating keratinocytes and in the cytoplasm of poorly differentiated tumor cells. Ki-67 index is higher in epithelial dysplasias and OSCC than in morphologically normal oral epithelium.     

Intravascular large B-cell lymphoma: report of three cases and analysis of the mTOR pathway

Intravascular large B-cell lymphoma (IVLBCL) is a rare, aggressive and often fatal non-Hodgkin lymphoma characterized by preferential growth of malignant B-cells within the lumina of small vessels. Rituximab plus anthracycline-based chemotherapy is the current standard regimen for IVLBCL, however it has minimal efficacy in relapsed or refractory diseases. Recent clinical trials have shown a significant anti-lymphoma activity of mammalian target of rapamycin (mTOR) inhibitors in relapsed and refractory diffuse large B-cell lymphoma (DLBCL); however, the activation status of the mTOR pathway and the therapeutic potential of mTOR inhibitors in IVLBCL have not yet been studied. Here we described the clinicopathological features of 3 cases of IVLBCL diagnosed at our institutions, and evaluated the activation status of the mTOR signaling in these tumors. Our results showed that the mTOR complex 2 pathway was selectively upregulated in IVLBCL, as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) with concomitant overexpression of nuclear p-Akt (Ser473) and vascular endothelial growth factor (VEGF)-A in the lymphoma cells. These data suggest that overactivation of mTOR pathway may play a role in lymphomagenesis of IVLBCL and mTORC2 inhibitors may be beneficial in treating IVLBCL.     

mTOR复合物在糖尿病肾病小鼠肾组织中的不同分布和表达

目的探讨哺乳动物雷帕霉素靶分子复合物(mTORC)在糖尿病肾病(DN)小鼠肾组织中的分布、表达。方法 14只C57BL/6小鼠随机分成对照组和DN组,每组各7只。DN组小鼠予以链脲菌素腹腔注射建立小鼠DN模型,采用生化技术检测小鼠血、尿肌酐以及白蛋白水平,组织学染色检测肾脏病理变化,免疫荧光以及免疫印迹技术检测肾组织中mTOR、mTOR第2448位丝氨酸磷酸化修饰后mTOR(p-mTOR)、mTORC1(Raptor)、mTORC2(Rictor)以及mTOR信号通路下游的效应蛋白磷酸化S6K1(p-S6K1)的分布和表达。结果 DN组小鼠血糖、尿白蛋白/肌酐比值明显增加(P0.01),肾小球明显增大(P0.05)。mTOR、Raptor以及Rictor在正常以及DN小鼠肾皮质和髓质中均有表达,主要表达在肾小球系膜区、毛细血管袢、皮质近曲小管以及外髓和内髓集合管上皮细胞中。其中正常小鼠内髓肾组织中未见p-S6K1表达,正常以及DN小鼠肾小球中未见p-mTOR表达。免疫印迹检测表明,DN小鼠肾组织中mTOR、p-mTOR、Raptor、Rictor以及p-S6K1均明显上升(P0.05)。结论 mTORC广泛分布于小鼠肾组织且参与DN的发生发展,但mTOR第2448位丝氨酸磷酸化并不直接参与高血糖介导的肾小球损伤。     

Expression of phosphorylated mTOR and its clinical significances in small cell lung cancer

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating the cell cycle and protein synthesis, and is an attractive molecule for novel molecular targeting therapy in various cancers, including non-small cell lung cancer (NSCLC). In contrast with NSCLC, mTOR expression has not been fully investigated in SCLC. In this study, we evaluated the correlations between mTOR expression and clinical characteristics in SCLC. Immunohistochemical staining for phosphorylated mTOR (p-mTOR) was performed and histoscores were calculated on 115 SCLC tissue specimens. Based on the distribution of the data, a histoscore of 60 was used as a cutoff to dichotomize SCLCs into low versus high expression groups. Extended-stage SCLCs showed significantly lower p-mTOR expression than those of a limited-stage (P = 0.008). Lymph node metastasis was more frequently detected in the low than high expression group (P = 0.074). The high p-mTOR expression group had a weak tendency toward prolonged overall survival, but the difference was not statistically significant (P = 0.170). We found that there is a significant difference in p-mTOR expression between different clinical stages in SCLC. This result indicates that p-mTOR might play a more pivotal role in the biologic behavior of early SCLCs than advanced ones and the effectiveness of mTOR inhibitors might vary according to the extent of disease.     

Respiratory syncytial virus induces phosphorylation of mTOR at ser2448 in CD8 T cells from nasal washes of infected infants

Respiratory syncytial virus (RSV)‐specific CD8⁺ T cell responses do not protect against reinfection. Activation of mammalian target of rapamycin (mTOR) impairs memory CD8⁺ T cell differentiation. Our hypothesis was that RSV inhibits the formation of CD8⁺ T cells memory responses through mTOR activation. To explore this, human and mouse T cells were used. RSV induced mTOR phosphorylation at Ser2448 in CD8 T cells. mTOR activation by RSV was completely inhibited using rapamycin. RSV‐infected children presented higher mTOR gene expression on nasal washes comparing to children infected with metapneumovirus and rhinovirus. In addition, RSV‐infected infants presented a higher frequency of CD8⁺ pmTORser2448⁺ T cells in nasal washes compared to RSV‐negative infants. Rapamycin treatment increased the frequency of mouse CD8 RSV‐M_282–90 pentamer‐positive T cells and the frequency of RSV‐specific memory T cells precursors. These data demonstrate that RSV is activating mTOR directly in CD8 T cells, indicating a role for mTOR during the course of RSV infection.     

Phosphorylated mTOR expression correlates with poor outcome in early-stage triple negative breast carcinomas

The mammalian target of rapamycin (mTOR) plays an important role in cell growth, proliferation, and metabolism. Some studies have associated phosphorylated mTOR (p-mTOR) expression with worse outcome in breast cancers. However, the significance of p-mTOR expression specifically in triple negative breast carcinoma (TNBC) is unknown. In this study, p-mTOR expression was evaluated by immunohistochemistry in 172 TNBCs and the result was correlated with clinicopathologic variables and disease outcome. The majority of tumors (72.1%) were p-mTOR positive; p-mTOR expression did not correlate with age, tumor size, grade, lymph node status, or tumor stage. In patients at stage 1 and 2 disease, those with p-mTOR expression had significantly worse overall as well as recurrence-free survival compared to those without p-mTOR expression. p-mTOR expression appears to be an adverse prognostic indicator in early-stage TNBCs. The assessment of p-mTOR expression in these tumors may also help to stratify patients for future target therapy studies.     

Therapeutic strategy targeting the mTOR–HIF-1α–VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia-inducible factor-1α (HIF-1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF-1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti-cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated-mTOR (p-mTOR), HIF-1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF-1α and VEGF between SEA and CLA, but it was noted that p-mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG-1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p-mTOR, HIF-1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p-mTOR, HIF-1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR-targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p-mTOR.     

mTOR/p70s6k参与巨核细胞多倍体化调控

目的:研究巨核细胞多倍体细胞周期调控的机制。方法:Western blot分析多倍体细胞模型中mTOR/p70s6k通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析S6K1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,mTOR表达增加及第2448位丝氨酸位点磷酸化,发生在G1期进入S期,S6K1的第421位苏氨酸/第424位丝氨酸位点磷酸化发生在G2/M期。结论:mTOR/S6K1通路参与巨核细胞多倍体细胞周期的调控。