PD-L1 expression correlates with tumor-infiltrating lymphocytes and better prognosis in patients with HPV-negative head and neck squamous cell carcinomas.

INTRODUCTION: The importance of immune tumor microenvironment in the prognosis of patients with head and neck squamous carcinomas (HNSCC) is increasingly recognized. We analyzed the prognostic relevance of PD-L1 and PD-1 expressions in relation to the infiltration by CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). METHODS: Samples from 372 surgically treated HPV-negative HNSCC patients were evaluated by immunohistochemistry for PD-L1 expression [both tumor proportion score (TPS) and combined proportion score (CPS)], PD-1 expression in immune cells, and density of infiltrating CD8+ and FOXP3+ TILs. PD-L1 expression and CD8+ TIL density were combined to establish the type of tumor microenvironment. RESULTS: 29.5% cases exhibited PD-L1 TPS positivity (≥ 1%), whereas PD-L1 CPS positivity (≥ 1%) was observed in 40% cases. 47.5% cases showed positive PD-1 expression (≥ 1%). PD-L1 and PD-1 positivity correlated with a high density of both CD8+ and FOXP3+ TILs. In univariate analysis, PD-L1 TPS positivity (P = 0.026), PD-L1 CPS positivity (P = 0.004), high density of CD8+ TIL (P = 0.001), and high density of FOXP3+ TIL (P = 0.004) were associated with a better disease-specific survival (DSS). However, in multivariate analysis, only high density of CD8+ TIL was associated with a better DSS (P = 0.002). The type of tumor microenvironment correlated with DSS (P = .008), with the better DSS observed in cases with type I (PD-L1 CPS positivity and high density of CD8+ TIL). CONCLUSIONS: High infiltration by CD8+ TIL is associated with better survival outcomes. Positive PD-L1 expression correlates with a high infiltration by TILs, explaining its association with better prognosis.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

Identification of mutations associated with acquired resistance to sunitinib in renal cell cancer.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

5 protein-based signature for resectable lung squamous cell carcinoma improves the prognostic performance of the TNM staging.

INTRODUCTION: Prognostic biomarkers have been very elusive in the lung squamous cell carcinoma (SCC) and none is currently being used in the clinical setting. We aimed to identify and validate the clinical utility of a protein-based prognostic signature to stratify patients with early lung SCC according to their risk of recurrence or death. METHODS: Patients were staged following the new International Association for the Study of Lung Cancer (IASLC) staging criteria (eighth edition, 2018). Three independent retrospective cohorts of 117, 96 and 105 patients with lung SCC were analysed to develop and validate a prognostic signature based on immunohistochemistry for five proteins. RESULTS: We identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (p<0.001; HR=4.06, 95% CI 2.18 to 7.56) and overall survival (OS) (p=0.004; HR=2.38, 95% CI 1.32 to 4.31). The prognostic capability of PI was confirmed in an external multi-institutional cohort for DFS (p=0.042; HR=2.01, 95% CI 1.03 to 3.94) and for OS (p=0.031; HR=2.29, 95% CI 1.08 to 4.86). Moreover, PI added complementary information to the newly established IASLC TNM 8th edition staging system. A combined prognostic model including both molecular and anatomical (TNM) criteria improved the risk stratification in both cohorts (p<0.05). CONCLUSION: We have identified and validated a clinically feasible protein-based prognostic model that complements the updated TNM system allowing more accurate risk stratification. This signature may be used as an advantageous tool to improve the clinical management of the patients, allowing the reduction of lung SCC mortality through a more accurate knowledge of the patient's potential outcome.

A novel protein-based prognostic signature improves risk stratification to guide clinical management in early-stage lung adenocarcinoma patients.

Each of the pathological stages (I-IIIa) of surgically resected non-small-cell lung cancer has hidden biological heterogeneity, manifested as heterogeneous outcomes within each stage. Thus, the finding of robust and precise molecular classifiers with which to assess individual patient risk is an unmet medical need. Here, we identified and validated the clinical utility of a new prognostic signature based on three proteins (BRCA1, QKI, and SLC2A1) to stratify early-stage lung adenocarcinoma patients according to their risk of recurrence or death. Patients were staged according to the new International Association for the Study of Lung Cancer (IASLC) staging criteria (8th edition, 2018). A test cohort (n = 239) was used to assess the value of this new prognostic index (PI) based on the three proteins. The prognostic signature was developed by Cox regression with the use of stringent statistical criteria (TRIPOD: Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis). The model resulted in a highly significant predictor of 5-year outcome for disease-free survival (p < 0.001) and overall survival (p < 0.001). The prognostic ability of the model was externally validated in an independent multi-institutional cohort of patients (n = 114, p = 0.021). We also demonstrated that this molecular classifier adds relevant information to the gold standard TNM-based pathological staging, with a highly significant improvement of the likelihood ratio. We subsequently developed a combined PI including both the molecular and the pathological data that improved the risk stratification in both cohorts (p ≤ 0.001). Moreover, the signature may help to select stage I-IIA patients who might benefit from adjuvant chemotherapy. In summary, this protein-based signature accurately identifies those patients with a high risk of recurrence and death, and adds further prognostic information to the TNM-based clinical staging, even when the new IASLC 8th edition staging criteria are applied. More importantly, it may be a valuable tool for selecting patients for adjuvant therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.

The oncogenic RNA-binding protein SRSF1 regulates LIG1 in non-small cell lung cancer.

In recent years, the relevance of RNA metabolism has been increasingly recognized in a variety of diseases. Modifications in the levels of RNA-binding proteins elicit changes in the expression of cancer-related genes. Here we evaluate whether SRSF1 regulates the expression of DNA repair genes, and whether this regulation has a relevant role in lung carcinogenesis. An in silico analysis was performed to evaluate the association between the expression of SRSF1 and DNA repair genes. In vitro functional analyses were conducted in SRSF1 or DNA ligase 1 (LIG1)-downregulated non-small cell lung cancer (NSCLC) cell lines. In addition, the prognostic value of LIG1 was evaluated in NSCLC patients by immunohistochemistry. We found a significant correlation between the DNA repair gene LIG1 and SRSF1 in NSCLC cell lines. Moreover, SRSF1 binds to LIG1 mRNA and regulates its expression by increasing its mRNA stability and enhancing its translation in an mTOR-dependent manner. Furthermore, siRNA-mediated LIG1 inhibition reduced proliferation and increased apoptosis of NSCLC cells. Finally, the expression of LIG1 was an independent prognostic factor for NSCLC, as confirmed in a series of 210 patients. These results show that LIG1 is regulated by the oncoprotein SRSF1 and plays a relevant role in lung cancer cell proliferation and progression. LIG1 is associated with poor prognosis in non-small lung cancer patients.

The sVEGFR1-i13 splice variant regulates a ß1 integrin/VEGFR autocrine loop involved in the progression and the response to anti-angiogenic therapies of squamous cell lung carcinoma.

BACKGROUND: While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known. METHODS: mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples. RESULTS: We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/ß1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and ß1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC. CONCLUSIONS: Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies.

Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations.

BACKGROUND: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. RESULTS: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). CONCLUSION: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.

Phosphorylated tubulin adaptor protein CRMP-2 as prognostic marker and candidate therapeutic target for NSCLC.

Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.

Receptor of activated protein C promotes metastasis and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma.

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.

DSTYK inhibition increases the sensitivity of lung cancer cells to T cell-mediated cytotoxicity.

Lung cancer remains the leading cause of cancer-related death worldwide. We identify DSTYK, a dual serine/threonine and tyrosine non-receptor protein kinase, as a novel actionable target altered in non-small cell lung cancer (NSCLC). We also show DSTYK's association with a lower overall survival (OS) and poorer progression-free survival (PFS) in multiple patient cohorts. Abrogation of DSTYK in lung cancer experimental systems prevents mTOR-dependent cytoprotective autophagy, impairs lysosomal biogenesis and maturation, and induces accumulation of autophagosomes. Moreover, DSTYK inhibition severely affects mitochondrial fitness. We demonstrate in vivo that inhibition of DSTYK sensitizes lung cancer cells to TNF-α-mediated CD8+-killing and immune-resistant lung tumors to anti-PD-1 treatment. Finally, in a series of lung cancer patients, DSTYK copy number gain predicts lack of response to the immunotherapy. In summary, we have uncovered DSTYK as new therapeutic target in lung cancer. Prioritization of this novel target for drug development and clinical testing may expand the percentage of NSCLC patients benefiting from immune-based treatments.

SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation.

INTRODUCTION: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. METHODS: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. RESULTS: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. CONCLUSIONS: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer.

BACKGROUND: Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. RESULTS: The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. CONCLUSIONS: This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A.

BACKGROUND: Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. RESULTS: Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found. CONCLUSIONS: Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.

TGFBI expression is associated with a better response to chemotherapy in NSCLC.

BACKGROUND: Lung cancer is one of the most prevalent neoplasias in developed countries. Advances in patient survival have been limited and the identification of prognostic molecules is needed. Resistance to treatment is strongly related to tumor cell adhesion to the extracellular matrix and alterations in the quantity and nature of molecules constituting the tumor cell niche. Recently, transforming growth factor beta-induced protein (TGFBI), an extracellular matrix adaptor protein, has been reported to be differentially expressed in transformed tissues. Loss of TGFBI expression has been described in several cancers including lung carcinoma, and it has been suggested to act as a tumor suppressor gene. RESULTS: To address the importance of TGFBI expression in cancer progression, we determined its expression in NSCLC clinical samples using immunohistochemistry. We identified a strong association between elevated TGFBI expression and the response to chemotherapy. Furthermore, we transiently over-expressed and silenced TGFBI in human NSCLC cell lines. Cells over-expressing TGFBI displayed increased sensitivity to etoposide, paclitaxel, cisplatin and gemcitabine. We observed that TGFBI-mediated induction of apoptosis occurred through its binding to alphavbeta3 integrin. We also determined that full-length TGFBI did not induce caspase 3/7 activation but its proteolytic fragments that were < 3 kDa in size, were able to activate caspase 3, 7 and 8. This pro-apoptotic effect was blocked by anti-alphavbeta3 integrin antibodies. CONCLUSIONS: The results shown here indicate that TGFBI is a predictive factor of the response to chemotherapy, and suggest the use of TGFBI-derived peptides as possible therapeutic adjuvants for the enhancement of responses to chemotherapy.

microRNA-451 regulates macrophage migration inhibitory factor production and proliferation of gastrointestinal cancer cells.

PURPOSE: microRNAs (miRNA) are small RNAs that function as post-transcriptional regulators of gene expression. Recent evidence has shown that some miRNAs can act as oncogenes or tumor suppressors. This study was conducted to evaluate the potential association of miRNA expression with clinical outcome in patients with gastric cancer. EXPERIMENTAL DESIGN: Expression of 250 human mature miRNAs was measured by real-time PCR on paraffin-embedded tumor samples of 21 patients with gastric cancer stage III uniformly treated with surgical resection followed by chemoradiation. We identified the miRNAs correlated with disease-free and overall survival times, and the results were evaluated including 24 other patients. In vitro cell proliferation and radiosensitivity studies were done to support clinical data. RESULTS: The results revealed that down-regulation of miR-451 was associated with worse prognosis. miR-451 was detected by in situ hybridization in epithelial cells and showed decreased expression in gastric and colorectal cancer versus nontumoral tissues. Overexpression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. Microarray and bioinformatic analysis identified the novel oncogene macrophage migration inhibitory factor (MIF) as a potential target of miR-451. In fact, overexpression of miR-451 down-regulated mRNA and protein levels of MIF and decreased expression of reporter genes with MIF target sequences. Moreover, we found a significant inverse correlation between miR-451 and MIF expression in tumoral gastric biopsies. CONCLUSIONS: These findings support the role of miR-451 as a regulator of cancer proliferation and open new perspectives for the development of effective therapies for chemoradioresistant cancers.

The SRC Inhibitor Dasatinib Induces Stem Cell-Like Properties in Head and Neck Cancer Cells that are Effectively Counteracted by the Mithralog EC-8042.

The frequent dysregulation of SRC family kinases (SFK) in multiple cancers prompted various inhibitors to be actively tested in preclinical and clinical trials. Disappointingly, dasatinib and saracatinib failed to demonstrate monotherapeutic efficacy in patients with head and neck squamous cell carcinomas (HNSCC). Deeper functional and mechanistic knowledge of the actions of these drugs is therefore needed to improve clinical outcome and to develop more efficient combinational strategies. Even though the SFK inhibitors dasatinib and saracatinib robustly blocked cell migration and invasion in HNSCC cell lines, this study unveils undesirable stem cell-promoting functions that could explain the lack of clinical efficacy in HNSCC patients. These deleterious effects were targeted by the mithramycin analog EC-8042 that efficiently eliminated cancer stem cells (CSC)-enriched tumorsphere cultures as well as tumor bulk cells and demonstrated potent antitumor activity in vivo. Furthermore, combination treatment of dasatinib with EC-8042 provided favorable complementary anti-proliferative, anti-invasive, and anti-CSC functions without any noticeable adverse interactions of both agents. These findings strongly support combinational strategies with EC-8042 for clinical testing in HNSCC patients. These data may have implications on ongoing dasatinib-based trials.

Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens.

BACKGROUND: The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. RESULTS: We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |Delta Ct| (= |Ct Normal-Ct tumor|) +/- SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |Delta Ct| (0.70 +/- 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability. CONCLUSION: Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.