Low-dose radiotherapy in diffuse large B-cell lymphoma.

Low-dose radiotherapy (LDRT) given in 2 × 2 Gy is a highly effective and safe treatment for palliation of indolent lymphomas. Otherwise, very little regarding the use of LDRT for diffuse large B-cell lymphoma (DLBCL) has been investigated. We designed a phase 2 trial of LDRT in patients with DLBCL with indication for palliative radiation. Low-dose radiotherapy was administered on symptomatic areas only. Clinical response was assessed 21 days after LDRT and defined as reduction >50% of maximum diameter of the radiated lesions. Quality of life was scored by the European Organisation for Research and Treatment of Cancer QLQ-C30 questionnaire. Tumor subtype (germinal center B-cell type versus activated B-cell type) and the presence of TP53 mutations in pathologic specimens of the target lesion were also evaluated. Twenty-three of twenty-five radiated patients were evaluable for response, and 2 died of disease before the visit at 21 days. The overall response rate was 70% (16 of 23 patients), with 7 complete responses and 9 partial responses (mean duration of response, 6 months; range, 1-39 months). Fifteen patients answered to the QLQ-C30 questionnaires, and an improved quality of life was documented in 9 cases. TP53 mutations were detected in 2 of 6 (33%) nonresponders and in none of the responders (P = .12). Germinal center B-cell type responded better than activated B-cell type (response rate was 83% and 29%, respectively, P = .01). These findings indicate that LDRT is effective for palliation in patients with DLBCL.

miR-135a Inhibits Cancer Stem Cell-Driven Medulloblastoma Development by Directly Repressing Arhgef6 Expression.

microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.

Transformation of normal human cells in the absence of telomerase activation.

Our knowledge of the transformation process has emerged largely from studies of primary rodent cells and animal models. However, numerous attempts to transform human cells using oncogene combinations that are effective in rodents have proven unsuccessful. These findings strongly argue for the study of homologous experimental systems. Here we report that the combined expression of adenovirus E1A, Ha-RasV12, and MDM2 is sufficient to convert a normal human cell into a cancer cell. Notably, transformation did not require telomerase activation. Therefore, we provide evidence that activation of telomere maintenance strategies is not an obligate characteristic of tumorigenic human cells.

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.

The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K-Akt Pathways.

The targeting of the Mitogen-Activated Protein Kinase (MAPK) signalling pathway in melanoma improves the prognosis of patients harbouring the V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutation. However, a fraction of these patients may experience tumour progression due to resistance to targeted therapy. Mutations affecting the Phosphoinositol-3-Kinase (PI3K)-Akt pathway may favour the onset of drug resistance, suggesting the existence of a crosstalk between the MAPK and PI3K-Akt pathways. We hypothesized that the inhibition of both pathways may be a therapeutic option in resistant melanoma. However, conflicting data have been generated in this context. In this study, three different A375 cell melanoma models either overexpressing or not expressing the wild-type or mutated form of the PhosphatidylInositol-4,5-bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) gene were used to clarify the therapeutic response of melanoma to BRAF, Mitogen-Activated Protein Kinase Kinase 1 (MEK), and PI3K inhibitors in the presence of the PIK3CA H1047R mutation. Our data strongly support the notion that the crosstalk between the MAPK and PI3K-Akt pathways is one of the main mechanisms associated with melanoma development and progression and that the combination of MAPK and PI3K inhibitors may sensitize melanoma cells to therapy.

Interference of p53:Twist1 interaction through competing nanobodies.

Twist1 promote the bypass of p53 response by interacting with p53 and facilitating its MDM2-mediated degradation. We reasoned that reagents able to interfere with the p53:Twist1 complex might alleviate Twist1 inhibitory effect over p53, thus representing potential therapeutic tools in p53 wild type tumors. From a pre-immune library of llama nanobodies (VHH), we isolated binders targeting the p53 C-terminal region (p53-CTD) involved in the interaction with Twist1 by using recombinant Twist1 as an epitope-specific competitor during elution. Positive hits were validated by proving their capacity to immunoprecipitate p53 and to inhibit Twist1:p53 binding in vitro. Molecular modeling confirmed a preferential docking of positive hits with p53-CTD. D11 VHH activity was validated in human cell models, succeeded in immunoprecipitating endogenous p53 and, similarly to Twist1 knock-down, interfered with p53 turnover, p53 phosphorylation at Serine 392 and affected cell viability. Despite the limited functional effect determined by D11 expression in target cells, our results provide the proof of principle that nanobodies ectopically expressed within a cell, have the capacity to target the assembly of the pro-tumorigenic Twist1:p53 complex. These results disclose novel tools for dissecting p53 biology and lay down the grounds for the development of innovative targeted therapeutic approaches.

Constitutive overexpression of CDC25A in primary human mammary epithelial cells results in both defective DNA damage response and chromosomal breaks at fragile sites.

CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer development, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established a cell model in which CDC25A was constitutively overexpressed in hTERT-immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability.

Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cks1 proteins.

Retinoic acid (RA) arrests the growth of EBV-immortalized lymphoblastoid B cell lines (LCLs) by upregulating the cyclin-dependent kinase inhibitor p27Kip1. Here, we show that in LCLs, RA inhibits ubiquitination and proteasome-dependent degradation of p27Kip1, a phenomenon that is associated with downregulation of Thr187 phosphorylation of the protein, whereas the phosphorylation on Ser10 is unaffected. Furthermore, we demonstrate that RA downregulates the expression of the p45Skp2 and Cks1 proteins, two essential components of the SCF(Skp2) ubiquitin ligase complex that target p27Kip1 for degradation. Downregulation of p45Skp2)and Cks1 occurs before the onset of growth arrest and is due to enhanced proteasome-mediated proteolysis of these proteins. Moreover, overexpression of p45Skp2 in DG75 cells prevents p27Kip1 protein accumulation and promotes resistance to the antiproliferative effects of RA. Treatment with Leptomycin B (LMB) blocked the translocation of p27Kip1 to the cytoplasm and prevented its degradation, indicating that CRM1-dependent nuclear export is required for p27Kip1 degradation. The shuttle protein p38Jab1, however, does not accumulate in the nucleus upon LMB treatment, nor does it interact with p27Kip1. Conversely, p45Skp2 is associated with p27Kip1 both in the nucleus and in the cytoplasm, accumulating within the nuclei after exposure to LMB and co-localizing with the exportin CRM1, suggesting a possible involvement of p45Skp2 in CRM1-dependent nuclear export of p27Kip1. These results indicate that downregulation of p45Skp2 is a key element underlying RA-induced p27Kip1 stabilization in B cells, resulting in an impaired targeting of the protein to the ubiquitin-proteasome pathway and probably contributing to the nuclear accumulation of p27Kip1.

SMARCB1/INI1 genetic inactivation is responsible for tumorigenic properties of epithelioid sarcoma cell line VAESBJ.

Epithelioid sarcoma is a rare soft tissue neoplasm that usually arises in the distal extremities of young adults. Epithelioid sarcoma presents a high rate of recurrences and metastases and frequently poses diagnostic dilemmas. We previously reported loss of tumor suppressor SMARCB1 protein expression and SMARCB1 gene deletion in the majority of epithelioid sarcoma cases. Unfortunately, no appropriate preclinical models of such genetic alteration in epithelioid sarcoma are available. In the present report, we identified lack of SMARCB1 protein due to a homozygous deletion of exon 1 and upstream regulatory region in epithelioid sarcoma cell line VAESBJ. Restoration of SMARCB1 expression significantly affected VAESBJ cell proliferation, anchorage-independent growth, and cell migration properties, thus supporting the causative role of SMARCB1 loss in epithelioid sarcoma pathogenesis. We investigated the translational relevance of this genetic background in epithelioid sarcoma and showed that SMARCB1 ectopic expression significantly augmented VAESBJ sensitivity to gamma irradiation and acted synergistically with flavopiridol treatment. In VAESBJ, both activated ERBB1/EGFR and HGFR/MET impinged on AKT and ERK phosphorylation. We showed a synergistic effect of combined inhibition of these 2 receptor tyrosine kinases using selective small-molecule inhibitors on cell proliferation. These observations provide definitive support to the role of SMARCB1 inactivation in the pathogenesis of epithelioid sarcoma and disclose novel clues to therapeutic approaches tailored to SMARCB1-negative epithelioid sarcoma.

MEF2 is a converging hub for histone deacetylase 4 and phosphatidylinositol 3-kinase/Akt-induced transformation.

The MEF2-class IIa histone deacetylase (HDAC) axis operates in several differentiation pathways and in numerous adaptive responses. We show here that nuclear active HDAC4 and HDAC7 display transforming capability. HDAC4 oncogenic potential depends on the repression of a limited set of genes, most of which are MEF2 targets. Genes verified as targets of the MEF2-HDAC axis are also under the influence of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that affects MEF2 protein stability. A signature of MEF2 target genes identified by this study is recurrently repressed in soft tissue sarcomas. Correlation studies depicted two distinct groups of soft tissue sarcomas: one in which MEF2 repression correlates with PTEN downregulation and a second group in which MEF2 repression correlates with HDAC4 levels. Finally, simultaneous pharmacological inhibition of the PI3K/Akt pathway and of MEF2-HDAC interaction shows additive effects on the transcription of MEF2 target genes and on sarcoma cells proliferation. Overall, our work pinpoints an important role of the MEF2-HDAC class IIa axis in tumorigenesis.

CDC25A protein stability represents a previously unrecognized target of HER2 signaling in human breast cancer: implication for a potential clinical relevance in trastuzumab treatment.

The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In particular, transgenic expression of CDC25A cooperates with HER2 in promoting mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced tumorigenesis penetrance. On the basis of this evidence of a synergism between HER2 and the cell cycle regulator CDC25A in a mouse model of mammary tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast cancer and its possible implications in therapeutic response. HER2 status and CDC25A expression were assessed in 313 breast cancer patients and we found statistically significant correlation between HER2 and CDC25A (P = .007). Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and very aggressive phenotype was identified (P = .005). Importantly, our in vitro studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on cell growth and viability relied also on CDC25A expression and that such inhibition induces CDC25A down-regulation through phosphatidylinositol 3-kinase/protein kinase B pathway and DNA damage response activation. In line with this observation, we found a statistical significant association between CDC25A overexpression and trastuzumab-combined therapy response rate in two different HER2-positive cohorts of trastuzumab-treated patients in either metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P = .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and CDC25A that positively modulates HER2-targeted therapy response, suggesting that, in HER2-positive breast cancer patients, CDC25A overexpression affects trastuzumab sensitivity.

A "twist box" code of p53 inactivation: twist box: p53 interaction promotes p53 degradation.

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates its MDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

TWIST1 plays a pleiotropic role in determining the anaplastic thyroid cancer phenotype.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by chemoresistance, local invasion, and distant metastases. ATC is invariably fatal. OBJECTIVE: The aim was to study the role of TWIST1, a basic helix-loop-helix transcription factor, in ATC. DESIGN: Expression of TWIST1 was studied by immunohistochemistry and real-time PCR in normal thyroids and well-differentiated, poorly differentiated, and ATC. The function of TWIST1 was studied by RNA interference in ATC cells and by ectopic expression in well-differentiated thyroid carcinoma cells. RESULTS: ATCs up-regulate TWIST1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Knockdown of TWIST1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of TWIST1 in thyroid cells induced resistance to apoptosis and increased cell migration and invasion. CONCLUSIONS: TWIST1 plays a key role in determining malignant features of the anaplastic phenotype in vitro.

Overexpression of TWIST2 correlates with poor prognosis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumors with variable presentation and clinical behavior. Despite improvements in surgical and radiation therapy techniques, the 5-year survival rate has not improved significantly over the past decades. Thus, there is an urgent need to identify novel markers that may allow for the development of personalized therapeutic approaches. In the present study we evaluated the prognostic role of the expression of genes related to the induction of epithelial mesenchymal transition (EMT). To this aim, a consecutive series of 69 HNSCC were analyzed for the expression of TWIST1, TWIST2, SNAI1, SNAI2, E-Cadherin, N-Cadherin and Vimentin.TWIST1, TWIST2, SNAI1 and SNAI2 were significantly overexpressed in HNSCC, with TWIST2, SNAI1 and SNAI2 being more markedly increased in tumors compared to normal mucosae. The expression of TWIST1 and SNAI2 was associated with upregulation of mesenchymal markers, but failed to correlate with pathological parameters or clinical behaviour. In contrast, we found that upregulation of TWIST2, which was independent of the activation of a mesenchymal differentiation program, correlated with poor differentiation grade (p=0.016) and shorter survival (p=0.025), and identifies a subset of node-positive oral cavity/pharynx cancer patients with very poor prognosis (p less than 0.001). Overall our study suggests that the assessment of TWIST2 expression might help to stratify HNSCC patients for risk of disease progression, pointing to TWIST2 as a potential prognostic marker.

Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence.

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.

Alterations of beta-catenin pathway in non-melanoma skin tumors: loss of alpha-ABC nuclear reactivity correlates with the presence of beta-catenin gene mutation.

To determine the role of beta-catenin pathway in human skin carcinogenesis, 135 non-melanoma skin tumors were analyzed for beta-catenin expression and gene mutations. Intense nucleo-cytoplasmic immunoreactivity for C terminus beta-catenin antibodies was observed in all pilomatricomas and in single cases of trichoepithelioma and squamous cell carcinoma showing peculiar signs of matrical differentiation. Moderate increase of beta-catenin nuclear staining was detected in a significant proportion of basal cell carcinomas, Bowen disease, spiroadenomas, and occasionally also in squamous cell carcinomas, but in these neoplasms only a limited fraction of tumor cells accumulated beta-catenin. Molecular analysis revealed that beta-catenin gene mutations are a peculiar feature of skin tumors with matrical differentiation and correlate with a pattern of intense and diffuse beta-catenin nuclear expression. In contrast, adenomatous polyposis coli (APC) and AXIN2 mutations were not involved in skin tumorigenesis. Analysis of Wnt pathway revealed that TCF-1 and MITF-M were selectively induced in the tumor types harboring beta-catenin mutations, indicating that a Wnt/beta-catenin pathway involving TCF-1 and MITF-M is activated in these tumors. Interestingly, high expression levels of TCF-3 were found in basal cell carcinomas and spiroadenomas. TCF-3 is reported to act as a negative modulator of beta-catenin degradation pathway. Thus, the moderate increase of beta-catenin nuclear staining detected in these tumor types might, at least in part, be due to a TCF-3-dependent mechanism. Finally, we found that the presence of beta-catenin mutations significantly correlated with loss of nuclear immunoreactivity for an antibody raised against the N terminus of beta-catenin (alphaABC). Thus, a combined analysis with C terminus-beta-catenin antibodies and alphaABC Ab may represent a powerful investigative approach for the detection of beta-catenin structural alterations.