Helena Pópulo

Frequency of TERT promoter mutations in human cancers

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

The mTOR Signalling Pathway in Human Cancer

The conserved serine/threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin, activates S6K1 and 4EBP1, which are involved in mRNA translation. It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress signals, and essential signalling pathways, such as PI3K, MAPK and AMPK, in order to control cell growth, proliferation and survival. mTORC2 is considered resistant to rapamycin and is generally insensitive to nutrients and energy signals. It activates PKC-α and AKT and regulates the actin cytoskeleton. Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression) has been reported in many types of cancers, particularly in melanoma, where alterations in major components of the mTOR pathway were reported to have significant effects on tumour progression. Therefore, mTOR is an appealing therapeutic target and mTOR inhibitors, including the rapamycin analogues deforolimus, everolimus and temsirolimus, are submitted to clinical trials for treating multiple cancers, alone or in combination with inhibitors of other pathways. Importantly, temsirolimus and everolimus were recently approved by the FDA for the treatment of renal cell carcinoma, PNET and giant cell astrocytoma. Small molecules that inhibit mTOR kinase activity and dual PI3K-mTOR inhibitors are also being developed. In this review, we aim to survey relevant research, the molecular mechanisms of signalling, including upstream activation and downstream effectors, and the role of mTOR in cancer, mainly in melanoma.

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the “alternative mechanism of telomere lengthening” (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target.

mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma

CONTEXT There are several genetic and molecular evidences suggesting dysregulation of the mammalian target of rapamycin (mTOR) pathway in thyroid neoplasia. Activation of the phosphatidylinositol-3-kinase/AKT pathway by RET/PTC and mutant RAS has already been demonstrated, but no data have been reported for the BRAF(V600E) mutation. OBJECTIVE The aim of this study was to evaluate the activation pattern of the mTOR pathway in malignant thyroid lesions and whether it may be correlated with known genetic alterations, as well as to explore the mechanisms underlying mTOR pathway activation in these neoplasias. RESULTS We observed, by immunohistochemical evaluation, an up-regulation/activation of the mTOR pathway proteins in thyroid cancer, particularly in conventional papillary thyroid carcinoma (cPTC). Overactivation of the mTOR signaling was particularly evident in cPTC samples harboring the BRAF(V600E) mutation. Transfection assays with BRAF expression vectors as well as BRAF knockdown by small interfering RNA revealed a positive association between BRAF expression and mTOR pathway activation, which appears to be mediated by pLKB1 Ser428, and emerged as a possible mechanism contributing to the association between BRAF mutation and mTOR pathway up-regulation. When we evaluated the rapamycin in the growth of thyroid cancer cell lines, we detected that cell lines with activating mutations in the MAPK pathway show a higher sensitivity to this drug. CONCLUSIONS We determined that the AKT/mTOR pathway is particularly overactivated in human cPTC harboring the BRAF(V600E) mutation. Moreover, our results suggest that the mTOR pathway could be a good target to enhance therapy effects in certain types of thyroid carcinoma, namely in those harboring the BRAF(V600E) mutation.

TERT Promoter Mutations in Skin Cancer: The Effects of Sun Exposure and X-Irradiation

The reactivation or reexpression of telomerase (TERT) is a widespread feature of neoplasms. TERT promoter mutations were recently reported that were hypothesized to result from UV radiation. In this retrospective study, we assessed TERT promoter mutations in 196 cutaneous basal cell carcinomas (BCCs), including 102 tumors from X-irradiated patients, 94 tumors from patients never exposed to ionizing radiation treatment, and 116 melanomas. We sought to evaluate the effects of UV and X-ray irradiation on TERT mutation frequency. TERT mutations were detected in 27% of BCCs from X-irradiated patients, 51% of BCCs from nonirradiated patients, and 22% of melanoma patients. TERT mutations were significantly increased in non-X-irradiated BCC patients compared with X-irradiated BCC patients; the mutations also presented a different mutation signature. In nonirradiated patients, TERT mutations were more frequent in BCCs of sun-exposed skin, supporting a possible causative role of UV radiation. In melanoma, TERT promoter mutations were generally restricted to intermittent sun-exposed areas and were associated with nodular and superficial spreading subtypes, increased thickness, ulceration, increased mitotic rate, and BRAFV600E mutations. Our results suggest that various carcinogenic factors may cause distinct TERT promoter mutations in BCC and that TERT promoter mutations might be associated with a poorer prognosis in melanoma.

Evaluation of the mTOR pathway in ocular (uvea and conjunctiva) melanoma.

Ocular melanoma is the most common eye malignancy in adults. It usually arises in the uvea, mostly in the choroid and less frequently in the conjunctiva. There is no curative therapy available when it becomes metastatic. The etiopathogenesis of uvea and conjunctiva melanomas is still poorly understood. The mammalian target of rapamycin (mTOR) pathway is involved in many biological processes and has been implicated in the development of cutaneous melanoma tumours. The mTOR pathway is an important target for anticancer drug development, and an inhibitor of this pathway has already been approved for use in humans to treat advanced renal cell carcinoma. The aim of this study was to evaluate the contribution of the mTOR pathway in uvea and conjunctiva melanomas. We analysed specific mTOR pathway effectors using immunohistochemical analysis of 30 uvea and eight conjunctiva melanoma samples. We assessed the association with prognostic clinical-pathological features, and performed mutational analysis on the BRAF and NRAS genes. None of the cases had mutations in either BRAF or NRAS. Expression of phospho-AKT Thr308 was associated with metastatic uvea melanomas. In conjunctiva melanomas, overactivation of the mTOR pathway, as confirmed by high phospho-AKT Ser473 and Thr308, S6 and p4EBP1 Thr37/46 levels, was associated with adverse prognostic parameters (mitotic index and tumour thickness). Conjunctiva melanomas displayed high expression of phospho-mTOR effectors in contrast with uvea melanomas, in which PTEN seemed to downregulate the mTOR pathway. Characterizing the expression of PTEN, AKT and pS6 Ser235/236 might be a useful predictive tool for deciding whether to use mTOR inhibitors to treat conjunctiva melanomas.

Analysis of GNAQ mutations, proliferation and MAPK pathway activation in uveal melanomas

Aim To study the GNAQ mutational status in a series of uveal melanomas and evaluate possible associations with mitogen-activated protein kinase (MAPK) pathway protein expression and tumour proliferation markers. Methods Mutational analysis was performed by PCR/sequencing of exon 5 of the GNAQ gene in a series of 22 uveal melanomas in which total and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 overexpression without coexistent BRAF and NRAS mutations had previously been observed. Expression of the cell cycle markers (Ki-67, cyclin D1 and p27) was evaluated by immunohistochemistry. The association between GNAQ mutational status, ERK1/2, phospho-ERK1/2, Ki-67, cyclin D1 and p27 expression levels and the clinicopathological prognostic parameters of uveal melanomas was also assessed. Results GNAQ mutations were found in 36% of uveal melanomas. No associations were found between the GNAQ mutational status and prognostic parameters, the expression of ERK1/2, pERK1/2 and cell cycle markers. Conclusion The results of this study suggest that GNAQ mutated uveal melanomas do not exhibit a higher deregulation of proliferation or higher activation of the MAPK signalling pathway than uveal melanomas without GNAQ overactivation.

Insights into melanoma: targeting the mTOR pathway for therapeutics

Introduction: Cutaneous melanoma represents < 5% of all skin cancers, but is responsible for the majority of skin cancer-related deaths. Ocular melanoma is the most common primary eye tumor in adults, and accounts for approximately 5% of all melanomas. Despite new diagnostic and therapeutic tools, the overall survival of patients treated for melanoma has not improved and most patients die of metastatic disease. Therefore, clarification of the molecular mechanisms underlying the etiopathogenesis of cutaneous and ocular melanomas may help determining the prognosis and tailoring therapy of patients harboring melanomas. Areas covered: In this review the authors aim to survey relevant research in the molecular mechanisms underlying melanomagenesis, and therapies under evaluation with emphasis in the mTOR pathway. Expert opinion: Despite an increasingly understanding of the genetics and biochemistry of melanoma, the mechanisms underlying their complex interactions are still poorly understood. Their clarification will lead to more successful therapeutic strategies and evidence-based management of patients with melanoma. More active drug combinations together with appropriate melanoma patient stratification based on molecular biomarkers will be essential for new advances in melanoma therapy.

mTOR pathway activation in cutaneous melanoma is associated with poorer prognosis characteristics

Dear Editor, The conserved serine ⁄ threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K ⁄ AKT pathway, is a major regulator of survival, growth, proliferation and motility, in response to mitogens, energy and nutrient levels (reviewed in Guertin and Sabatini, 2007). mTOR is an attractive therapeutic target, and mTORC1 inhibitors, including the rapamycin analogues Deforolimus, Everolimus and Tensirolimus, alone or in combination with inhibitors of other pathways, are undergoing clinical trials for treating multiple cancers, including melanoma (reviewed in Dancey, 2006; Fecher et al., 2007). Alterations in molecules of the PI3K ⁄ AKT ⁄ mTOR pathway have been identified in cutaneous melanomas (reviewed in Hocker et al., 2008). In view of the possible involvement of the PI3K ⁄ AKT ⁄ mTOR pathway in melanoma, as well as its potential role as a therapeutic target, we aimed to clarify whether there is an association between the expression of the PI3K ⁄ AKT ⁄ mTOR pathway effectors, clinico-pathological and prognostic parameters, and BRAF and NRAS mutations in cutaneous melanomas. To evaluate mTOR pathway expression in cutaneous melanoma, tissue microarrays of 84 cutaneous melanomas cases (Supporting Information Table S1) were stained with antibodies against PI3K ⁄ AKT ⁄ mTOR and MAPK pathways effectors: PTEN, AKT, pAKT Ser473, pAKT Thr308, mTOR, pmTOR Ser2448, S6, pS6 Ser235 ⁄ 236, 4E-BP1, p4E-BP1 Thr37 ⁄ 46, ERK1 ⁄ 2 and pERK1 ⁄ 2 Thr202 ⁄ Tyr204 (methods detailed in Supporting Information Data S1 and Table S2). The results of examination of the stained sections were consistent with activation of the AKT ⁄ mTOR pathway in cutaneous melanoma, as we observed a high expression of its active forms in most of the cases studied (Figure 1). Similar conclusions were reported by Karbowniczek et al. (2008), who evaluated pS6 in a series of cutaneous melanomas. We found a significant positive association between pmTOR and the two phosphorylated forms of AKT – pAKT Ser473 (P < 0.01) and pAKT Thr308 (P = 0.03). pmTOR was also associated with the two downstream effectors of mTORC1 – pS6 (P = 0.01) and p4EBP1 (P < 0.01). A positive association was found between the staining level of pS6 and p4EBP1 (P = 0.01). Expression of pAKT Ser473 was positively associated with the two downstream effectors of mTORC1 – pS6 and p4EBP1 (P < 0.01). Moreover, we found that the two phosphorylated forms of AKT – pAKT Ser473 and pAKT Thr308 – were positively associated with pERK1 ⁄ 2 (P < 0.01 and P = 0.01, respectively). pmTOR expression was also positively associated with pERK1 ⁄ 2 (P = 0.04), along with its two effectors pS6 and p4EBP1 (P < 0.01 and P = 0.02, respectively). The positive correlations found between the expression of upstream and downstream effectors of the mTOR pathway suggest simultaneous activation of the signalling pathway in cutaneous melanoma. Also, the positive correlation observed between the expression of the phosphorylated forms of mTOR effectors and pERK1 ⁄ 2 suggests that the MAPK pathway is activated concurrently with the AKT ⁄ mTOR pathway in cutaneous melanoma, similar to the pattern seen by our group in ocular melanomas (Populo et al., 2010). The mean expression level of the studied markers was compared with the clinico-pathological parameters of the cases (Table 1). We found that expression of pAKT Ser473 was significantly elevated (P = 0.02) in tumours with more than 1 mm thick, and a borderline significant higher mean of pS6 was found in cases with epidermal ulceration (P = 0.05). A significantly higher mean expression of AKT (P = 0.04), pAKT Ser473 (P < 0.01) and pS6 (P = 0.02) was detected in cases with III ⁄ IV Clark level than in cases with I ⁄ II Clark level. pAKT Ser473, pmTOR and pS6 expression was also significantly elevated in tumours with a mitotic rate greater than 1 mitosis ⁄ 10 high-power fields (P = 0.01). The association found between the expression of several proteins of the mTOR pathway and a poorer prognosis suggests an association between mTOR pathway

GNAQ and BRAF mutations show differential activation of the mTOR pathway in human transformed cells

Somatic mutations in GNAQ gene were described as being the main oncogenic activation in uveal melanomas, whereas mutations in BRAF gene have been described as a key genetic alteration that contributes to skin melanoma development. We have previously reported differential activation of the MAPK and AKT/mTOR signalling pathways in uveal and skin melanomas harbouring, respectively, GNAQ and BRAF mutations. The aim of this work was to compare the functional effect of GNAQ and BRAF mutations in mTOR and MAPK pathway activation, cell proliferation and apoptosis. In this work, we performed transient transfection of HEK293 cells with BRAFWT, BRAFV 600E, GNAQWT, GNAQQ209P and GNAQQ209L vectors. We treated melanoma cell lines displaying different BRAF and GNAQ mutational status with the mTOR inhibitor RAD001 and with the MEK1/2 inhibitor U0126 and evaluated the effects in the growth of the cell lines and in mTOR and MAPK pathway effectors expression. At variance with the significant increase in the level of pmTOR Ser2448 and pS6 Ser235/236 proteins observed in cells transfected with BRAF vectors, no significant alteration in mTOR pathway effectors was observed in cells transfected with the three GNAQ expressing vectors. Also, GNAQ overexpression enhances Stat3 activation, which might mediate GNAQ oncogenic effects. None of the vectors led to significant differences in proliferation or apoptosis in the transfected cell lines. Cell lines harbouring a BRAF mutation were more sensitive to RAD001 treatment. U0126 leads to the reduction of MAPK and mTOR pathways activation in all cell lines tested. Our results indicate that GNAQ and BRAF activation drive distinct intracellular signalling pathways that may be useful for therapeutic decisions in human melanomas.