EDITORIAL Pathol. Oncol. Res., 14 February 2024 https://doi.org/10.3389/pore.2024.1611691

Cancer hypoxia, recognized as one of the most important hallmarks of cancer, affects gene expression, metabolism and ultimately tumor biology-related processes. Major causes of cancer hypoxia are deficient or inappropriate vascularization and systemic hypoxia of the patient (frequently induced by anemia), leading to a unique form of genetic reprogramming by hypoxia induced transcription factors (HIF). However, constitutive activation of oncogene-driven signaling pathways may also activate hypoxia signaling independently of oxygen supply. The consequences of HIF activation in tumors are the angiogenic phenotype, a novel metabolic profile and the immunosuppressive microenvironment. Cancer hypoxia and the induced adaptation mechanisms are two of the major causes of therapy resistance. Accordingly, it seems inevitable to combine various therapeutic modalities of cancer patients by existing anti-hypoxic agents such as anti-angiogenics, anti-anemia therapies or specific signaling pathway inhibitors. It is evident that there is an unmet need in cancer patients to develop targeted therapies of hypoxia to improve efficacies of various anti-cancer therapeutic modalities. The case has been opened recently due to the approval of the first-in-class HIF2α inhibitor.

Metastasis suppressor genes (MSGs) inhibit different biological processes during metastatic progression without globally influencing development of the primary tumor. The first MSG, NM23 (non-metastatic clone 23, isoform H1) or now called NME1 (stands for non-metastatic) was identified some decades ago. Since then, ten human NM23 paralogs forming two groups have been discovered. Group I NM23 genes encode enzymes with evolutionarily highly conserved nucleoside diphosphate kinase (NDPK) activity. In this review we summarize how results from NDPKs in model organisms converged on human NM23 studies. Next, we examine the role of NM23-H1 and its homologs within the metastatic cascade, e.g. cell migration and invasion, proliferation and apoptosis. NM23-H1 homologs are well known inhibitors of cell migration. Drosophila studies revealed that AWD, the fly counterpart of NM23-H1 is a negative regulator of cell motility by modulating endocytosis of chemotactic receptors on the surface of migrating cells in cooperation with Shibire/Dynamin; this mechanism has been recently confirmed by human studies. NM23-H1 inhibits proliferation of tumor cells by phosphorylating the MAPK scaffold, kinase suppressor of Ras (KSR), resulting in suppression of MAPK signalling. This mechanism was also observed with the C. elegans homolog, NDK-1, albeit with an inverse effect on MAPK activation. Both NM23-H1 and NDK-1 promote apoptotic cell death. In addition, NDK-1, NM23-H1 and their mouse counterpart NM23-M1 were shown to promote phagocytosis in an evolutionarily conserved manner. In summary, inhibition of cell migration and proliferation, alongside actions in apoptosis and phagocytosis are all mechanisms through which NM23-H1 acts against metastatic progression.

Several monoclonal antibodies and inhibitors targeting signalling pathways are being used in personalised medicine. Anti-EGFR antibodies seem to be effective, however, therapy resistance often occurs in colon carcinoma cases. mTOR inhibitors (mTORIs) could have a potential role in the breakthrough of therapy resistance. The mTOR activity related protein expression patterns and the in vitro effects of EGFR inhibitors (EGFRIs), mTORIs and their combinations were studied in different colon carcinoma cell lines (with different genetic backgrounds). Alamar Blue test and flow cytometry were used to analyse the in vitro proliferation and apoptotic effects of cetuximab, gefitinib, cisplatin, rapamycin, PP242 and NVP-BEZ235. The expressions of mTOR activity related proteins (p-70S6K, p-S6, Rictor, p-mTOR, Raptor) were studied by Western blot, immunocytochemistry and Duolink staining. The EGFRI resistance of the studied colon carcinoma cell lines related to their known mutations were confirmed, neither gefitinib nor cetuximab inhibited the proliferation or induced apoptosis in vitro. Individual differences in Rictor and Raptor expressions were detected by Western blot and immunocytochemistry beside elevated mTOR activity of these different colon carcinoma cell lines. These expression patterns correlated to the mTORIs sensitivity differences, moreover, mTORIs could enhance the effects of EGFRIs and other in vitro treatments. Our results suggest that mTORI combinations could be helpful in both EGFRI and platinum-based therapy of colon carcinomas. Moreover, we suggest determining both mTOR complex activity and mutations in Akt/mTOR signalling pathways for selecting the appropriate mTORIs and patients in potential future combination treatments.

Aims Tumour heterogeneity and altered activation of signalling pathways play important roles in therapy resistance. The PI3K/Akt/mTOR signalling network is a well-known regulator of several functions that contribute to tumour growth. mTOR exists in two functionally different multiprotein complexes. We aimed to determine mTOR activity-related proteins in clinically followed, conventionally treated colon carcinomas and to analyse the correlation between clinical data and mTORC1 and mTORC2 activity.Methods Immunohistochemistry was performed with different antibodies on tissue microarray blocks from 103 patients with human colorectal adenocarcinoma. mTORC1-and mTORC2-related activity were scored on different stainings including analysis of the expression of Raptor and Rictor-specific elements of mTORC1 and C2 complexes. The staining scores and clinical/survival data were compared and analysed.Results Detailed characterisation showed stage and grade independent high mTOR activity in 74% of cases. High mTOR activity was present in mTORC1 and/or mTORC2 complexes; > 60% of cases had mTORC2-related high mTOR activity. Based on our analysis, high mTOR activity and Rictor overexpression could be markers of a bad prognosis. Combined phosphoprotein and Rictor/Raptor expression evaluation revealed even stronger statistical correlation with prognosis.Conclusions The presented staining panel could be appropriate and highly recommended for the accurate specification of mTORC1 and C2 activity of tumour tissues. This could help in the selection of mTOR inhibitors and can provide information about prognosis, which may guide decisions about the intensity of therapy.

Brain metastases (BMs) are common complications of adenocarcinomas (ADCs) of the lung and are associated with a poor prognosis. Although an increasing amount of data indicates that dysregulated activity of mammalian target of rapamycin (mTOR) can influence the metastatic potential of various tumors, the role of mTOR complexes in the development of BMs from ADCs of the lung is largely unknown. To estimate mTOR activity, we studied the expression of mTOR-related proteins (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor) in primary (n=67) and brain metastatic (n=67) lung ADCs, including 15 paired tissue samples, using immunohistochemistry and tissue microarrays. Correlation with clinicopathological parameters was also analyzed. Increased p-mTOR, p-S6, and Rictor expressions were observed in 34%, 33%, and 37% of primary ADCs and in 79%, 70%, and 66% of BMs, respectively. Expression of these markers was significantly higher in BMs as compared with primary carcinomas (P<.0001, P<.0001, P<.001). Rictor expression was significantly higher in primary ADCs of the paired cases with BMs as compared with primary ADCs without BMs (67% versus 28%; P<.01). No other statistically significant correlations were found between mTOR activity and clinicopathological parameters. The increased mTORC1/C2 activity in a subset of pulmonary ADCs and the higher incidence of increased mTORC1/C2 activity in BMs suggest that the immunohistochemistry panel for characterizing mTOR activity and its potential predictive and prognostic role warrants further investigations.

In spite of the improved efficacy of therapy, it still fails in 15-20 % of childhood acute lymphoblastic leukemia (ALL) patients. Recently, altered expression of certain miRNAs (miRs) have been described in ALL with potential effect on prognosis. Presence of certain miRs (miRNA-16, -21, -24, -29b, -128b, -142-3p, -155, -223) was characterized in human lymphoma and leukemia cells by real-time PCR. Expression of miRs in pediatric ALL patients (n = 24) was measured before chemotherapy, at conventional response checkpoints and at relapse. Correlation between altered miR expression and response to prednisolone at day 8 of therapy and long term prognosis was statistically analysed. Overexpression of "oncomiR/inflammamiR"-21 - which is characteristic in different tumors-was missing in human ALL cells. However, higher expression of miR-128b and lower expression of miR-223 is generally characteristic for human ALL cell lines and ALL cells isolated from pediatric patients. Correlation was shown between miR-128b expression and prognosis, prednisolone response and survival data in childhood ALL. Expression of miR-128b and miR-223-both are leukemia specific-changed in parallel with percentage of bone marrow blasts in remission and during relapse. Therefore, we suggest that overexpression of miR-128b and downregulation of miR-223 shows a significant correlation with treatment response and prognosis in childhood ALL.

TGF-β1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-β1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-β signaling, and through these pathways it is able to enhance TGF-β induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-β1 combination on cell growth and on TGF-β and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-β1 induced apoptosis and restored TGF-β1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-β sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-β1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-β1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-β resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-β1.

Abstract There is increasing attention for PI3K/Akt/mTOR pathway as a potential target in tumor therapy. The high activity of mTOR is a characteristic feature of many tumors including hematological malignancies. However, the pathological characterisation of mTOR activity of lymphoid malignancies - especially the activity of different complexes – is still incomplete. We studied the mTOR activity related phospho-proteins and the elements of mTOR complexes by using different techniques (ELISA, Western blot, different immunohistochemical stainings, flow cytometry) depending on the tumor sample type (fresh/fixed human tissues, isolated human cells from blood or bone marrow, cell lines). The expression and activity results were compared in order to find correlation with clinical data or to mTOR inhibitor sensitivity in vitro. The proliferation and apoptotic effects after in vitro treatments were detected by Alamar Blue assay and flow cytometry. We established the in situ measurement for the amount of mTORC1, C2 complexes by Duolink stainings to compare the effects of different mTOR inhibitors to the mTORC1/C2 complex availability in lymphoma cell cultures. Our studies described high mTOR activity in all acute lymphoid leukemia (ALL) cases and in the majority of mantle cell lymphomas, anaplastic large cell lymphomas, Burkitt lymphomas, diffuse large B cell lymphomas (DLBCL), Hodgkin lymphomas (HL) and in certain cutan T lymphomas. Further characterisation of HLs, DLBCLs and ALL cases showed that high mTOR activity especially with the presence of mTORC2 is a sign of unfavorable prognosis. mTOR activity related to p-S6 and p-4EBP1 expression (ELISA and flow cytometry) at diagnosis was significantly higher in patients with poor prognosis in ALL cases. In DLBCL patient's the results showed that high mTOR activity with Rictor overexpression significantly correlated to the worse prognosis and shorter survival. We also detected the sign of high mTOR activity in more than 90% of HLs (HLs usually with favorable prognosis), however, in these cases Rictor was not found (the mTOR activity related to mTORC1). In the different lymphoma cell lines increased mTOR activity was detected and we could describe different Rictor, Raptor and mTOR-Rictor complex expression. Moreover, the inhibitor sensitivity of the cell lines and the different expression of the elements related to mTORC1 and C2 correlated. Our results confirmed the increased mTOR activity related to mTORC1 and/or C2 complexes in certain lymphomas and ALLs. According to these analyses, the activity and expression of mTOR complexes should have potential prognostic value and high importance before mTOR inhibitor treatment, especially in the cases with high mTORC2 related protein expression. The study was supported by OTKA816624, OTKA 84262 projects and Tumor Progression Research Group of Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University Citation Format: Anna Sebestyén, Noémi Nagy, Ágnes Márk, Anna Molnár, Titanilla Dankó, Melinda Hajdu, Mónika Tóth, Botond Timár, Mónika Csóka, László Kopper. mTOR C1/2 activities - related protein expression and its potential prognostic/therapeutic importance in certain lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B47.

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25-30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments.

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250 mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

Background Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete. Methods mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines. Results The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents. Conclusions Targeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response.

Denosumab is a perfect example on the targeted anticancer therapy. The inhibition of RANKL activity suppressed the osteoclasts' resorptive function and so prevented skeletal related events. This effect is useful not only against bone metastases, but also in the treatment of other diseases caused by bone loss. In different solid tumors with bone metastasis the quality of life also improved, although the overall survival usually showed no change. On the market the main competitors for denosumab are still the bisphosphonates (questions of costs and reimbursement are not discussed) and some potential new agents e.g. Src kinases (as dasatinib, saracatinib, bosutinib), cathepsin K inhibitors, (e.g. odanacatib), and new selective estrogen receptor modulators (e.g. bazedoxifene, lasofoxifene). Nevertheless, today denosumab is one of the most powerful agents in bone-saving area.

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases with several subtypes with differing responses to therapy. The standard initial therapy for DLBCL continues using regimens like R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), regardless of the subtypes. Targeted therapy offers a new approach to improve clinical responses. mTOR signaling has been shown to be activated in several lymphomas. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma (MCL) therapy and clinical trials are ongoing in other high grade lymphomas as well. However, there is limited data about mTOR activity in different DLBCLs. We evaluated immunohistochemical (IHC) results in biopsy materials from 79 DLBCL patients (42 females and 37 males, mean age 61 years), who were conventionally treated with R-CHOP, R-CEOP or CHOP therapy. Several antibodies (phospho-S6, phospho-p70S6K, phospho-mTOR, p-4EBP1, Mum1, CD10, bcl-6, bcl-2) were used and more than 700 IHC stainings were evaluated on TMA slides. Mum1, CD10 and bcl-6 staining helped to distinguish between germinal center (GC) and non-germinal center B-cell derived DLBCL cases. Approximately 30% of cases were characterized as GC-derived DLBCL, which showed virtually no mTOR activity, as determined by phosphorylated S6 (p-S6) expression, the most sensitive marker (only weak p-S6 expression was detected in 2 cases). However, in about 80% of non-GC lymphoma cases, p-S6, p-mTOR, p-p70S6K and p-4EBP1 positivity was observed, which alludes to mTOR activity. Although the importance of mTOR activity is well demonstrated by mTOR mediated cyclinD1 expression in MCL, no statistical correlation was found between mTOR activity and the expression of pro-proliferative or anti-apoptotic factors (such as bcl-2 and bcl-6) in the examined DLBCL cases. These results suggest that mTOR activity is characteristic in most non-GC derived DLBCLs. Similarly to MCL patients, those patients with poor survival may benefit from rapamycin derivatives or other treatments targeting mTOR. These rapalogs or other novel anti-mTOR agents may be used in addition to the regular R-CHOP therapy, however, patient selection criteria should be carefully considered. Supported by OTKA-K68341 and OTKA-K81624 Projects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2011-3163