ALK1 Loss Results in Vascular Hyperplasia in Mice and Humans Through PI3K Activation.

OBJECTIVE: ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-ß (transforming growth factor-ß) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia-a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia. APPROACH AND RESULTS: In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1+/- mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1+/- retinas and in turn normalize the vasculature. CONCLUSIONS: Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia.

TGFß Controls Ovarian Cancer Cell Proliferation.

There have been no major improvements in the overall survival of ovarian cancer patients in recent decades. Even though more accurate surgery and more effective treatments are available, the mortality rate remains high. Given the differences in origin and the heterogeneity of these tumors, research to elucidate the signaling pathways involved is required. The Transforming Growth Factor (TGFß) family controls different cellular responses in development and cell homeostasis. Disruption of TGFß signaling has been implicated in many cancers, including ovarian cancer. This article considers the involvement of TGFß in ovarian cancer progression, and reviews the various mechanisms that enable the TGFß signaling pathway to control ovarian cancer cell proliferation. These mechanistic explanations support the therapeutic use of TGFß inhibitors in ovarian cancer, which are currently in the early phases of development.

The TGFß pathway stimulates ovarian cancer cell proliferation by increasing IGF1R levels.

In a search for new therapeutic targets for treating epithelial ovarian cancer, we analyzed the Transforming Growth Factor Beta (TGFß) signaling pathway in these tumors. Using a TMA with patient samples we found high Smad2 phosphorylation in ovarian cancer tumoral cells, independently of tumor subtype (high-grade serous or endometrioid). To evaluate the impact of TGFß receptor inhibition on tumoral growth, we used different models of human ovarian cancer orthotopically grown in nude mice (OVAs). Treatment with a TGFßRI&II dual inhibitor, LY2109761, caused a significant reduction in tumor size in all these models, affecting cell proliferation rate. We identified Insulin Growth Factor (IGF)1 receptor as the signal positively regulated by TGFß implicated in ovarian tumor cell proliferation. Inhibition of IGF1R activity by treatment with a blocker antibody (IMC-A12) or with a tyrosine kinase inhibitor (linsitinib) inhibited ovarian tumoral growth in vivo. When IGF1R levels were decreased by shRNA treatment, LY2109761 lost its capacity to block tumoral ovarian cell proliferation. At the molecular level TGFß induced mRNA IGF1R levels. Overall, our results suggest an important role for the TGFß signaling pathway in ovarian tumor cell growth through the control of IGF1R signaling pathway. Moreover, it identifies anti-TGFß inhibitors as being of potential use in new therapies for ovarian cancer patients as an alternative to IGF1R inhibition.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism.

Phase II study of preoperative bevacizumab, capecitabine and radiotherapy for resectable locally-advanced rectal cancer.

BACKGROUND: To evaluate whether the addition of bevacizumab (BVZ) to capecitabine-based chemoradiotherapy in the preoperative treatment of locally advanced rectal cancer (LARC) improves efficacy measured by the pathological complete response (pCR) rate. METHODS: A phase II two-step design was performed. Patients received four cycles of therapy consisting of: BVZ 10 mg/kg in first infusion on day 1 and 5 mg/kg on days 15, 29, 43, capecitabine 1800 mg/m(2)/day 5 days per week during radiotherapy, which consisted of external-beam irradiation (45 Gy in 1.8 Gy dose per session over 5 sessions/week for 5 weeks). Six to eight weeks after completion of all therapies surgery was undergone. To profile the biological behaviour during BVZ treatment we measured molecular biomarkers before treatment, during BVZ monotherapy, and during and after combination therapy. Microvessel density (MVD) was measured after surgery. RESULTS: Forty-three patients were assessed and 41 were included in the study. Three patients achieved a pathological complete response (3/40: 7.5%) and 27 (67.5%) had a pathological partial response, (overall pathological response rate of 75%). A further 8 patients (20%) had stable disease, giving a disease control rate of 95%. Downstaging occurred in 31 (31/40: 77.5%) of the patients evaluated. This treatment resulted in an actuarial 4-year disease-free and overall survival of 85.4 and 92.7% respectively. BVZ with chemoradiotherapy showed acceptable toxicity. No correlations were observed between biomarker results and efficacy variables. CONCLUSION: BVZ with capecitabine and radiotherapy seem safe and active and produce promising survival results in LARC. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00847119 . Trial registration date: February 18, 2009.

New genetic drivers in hemorrhagic hereditary telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes are detected in around 90% of the patients; also 2% of patients harbor pathogenic variants at SMAD4 and GDF2. Importantly, the genetic cause of 8% of patients with clinical HHT remains unknown. Here, we present new putative genetic drivers of HHT. METHODS: To identify new HHT genetic drivers, we performed exome sequencing of 19 HHT patients and relatives with unknown HHT genetic etiology. We applied a multistep filtration strategy to catalog deleterious variants and prioritize gene candidates based on their known relevance in endothelial cell biology. Additionally, we performed in vitro validation of one of the identified variants. RESULTS: We identified variants in the INHA, HIF1A, JAK2, DNM2, POSTN, ANGPTL4, FOXO1 and SMAD6 genes as putative drivers in HHT. We have identified the SMAD6 p.(Glu407Lys) variant in one of the families; this is a loss-of-function variant leading to the activation of the BMP/TGFß signaling in endothelial cells. CONCLUSIONS: Variants in these genes should be considered for genetic testing in patients with HHT phenotype and negative for ACVRL1/ENG mutations.

Ovarian cancer relies on the PDGFRß-fibronectin axis for tumorsphere formation and metastatic spread.

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecological malignancy. The most common form of metastatic spread of HGSOC is transcoelomic dissemination. In this process, detached cells from the primary tumor aggregate as tumorspheres and promote the accumulation of peritoneal ascites. This represents an early event in HGSOC development and is indicative of poor prognosis. In this study, based on tumorspheres isolated from ascitic liquid samples from HGSOC patients, ovarian cancer spheroid 3D cultures, and in vivo models, we describe a key signal for tumorsphere formation in HGSOC. We report that platelet-derived growth factor receptor beta (PDGFRß) is essential for fibronectin-mediated cell clustering of ovarian cancer cells into tumorspheres. This effect is mediated by the kinase NUAK family SNF1-like kinase 1 (NUAK1) and blocked by PDGFRß pharmacological or genetic inhibition. In the absence of PDGFRß, ovarian cancer cells can be provided with fibronectin by cancer-associated fibroblasts to generate chimeric spheroids. This work provides new insights that uncover potential targets to prevent peritoneal dissemination, the main cause of advanced disease in HGSOC patients.

Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma.

Immune checkpoint blockade (ICB) approaches have changed the therapeutic landscape for many tumor types. However, half of cutaneous squamous cell carcinoma (cSCC) patients remain unresponsive or develop resistance. Here, we show that, during cSCC progression in male mice, cancer cells acquire epithelial/mesenchymal plasticity and change their immune checkpoint (IC) ligand profile according to their features, dictating the IC pathways involved in immune evasion. Epithelial cancer cells, through the PD-1/PD-L1 pathway, and mesenchymal cancer cells, through the CTLA-4/CD80 and TIGIT/CD155 pathways, differentially block antitumor immune responses and determine the response to ICB therapies. Accordingly, the anti-PD-L1/TIGIT combination is the most effective strategy for blocking the growth of cSCCs that contain both epithelial and mesenchymal cancer cells. The expression of E-cadherin/Vimentin/CD80/CD155 proteins in cSCC, HNSCC and melanoma patient samples predicts response to anti-PD-1/PD-L1 therapy. Collectively, our findings indicate that the selection of ICB therapies should take into account the epithelial/mesenchymal features of cancer cells.

Metronomic chemotherapy following the maximum tolerated dose is an effective anti-tumour therapy affecting angiogenesis, tumour dissemination and cancer stem cells.

In this article, the effectiveness of a multi-targeted chemo-switch (C-S) schedule that combines metronomic chemotherapy (MET) after treatment with the maximum tolerated dose (MTD) is reported. This schedule was tested with gemcitabine in two distinct human pancreatic adenocarcinoma orthotopic models and with cyclophosphamide in an orthotopic ovarian cancer model. In both models, the C-S schedule had the most favourable effect, achieving at least 80% tumour growth inhibition without increased toxicity. Moreover, in the pancreatic cancer model, although peritoneal metastases were observed in control and MTD groups, no dissemination was observed in the MET and C-S groups. C-S treatment caused a decrease in angiogenesis, and its effect on tumour growth was similar to that produced by the MTD followed by anti-angiogenic DC101 treatment. C-S treatment combined an increase in thrombospondin-1 expression with a decrease in the number of CD133+ cancer cells and triple-positive CD133+/CD44+/CD24+ cancer stem cells (CSCs). These findings confirm that the C-S schedule is a challenging clinical strategy with demonstrable inhibitory effects on tumour dissemination, angiogenesis and CSCs.

The impact of KRAS mutations on VEGF-A production and tumour vascular network.

BACKGROUND: The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. METHODS: Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. RESULTS: Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. CONCLUSION: Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis.

Effectivity of pazopanib treatment in orthotopic models of human testicular germ cell tumors.

BACKGROUND: Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs. METHODS: We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated. RESULTS: TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth. CONCLUSIONS: We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies.

Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway.

Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at -1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells.

Rethinking growth factors: the case of BMP9 during vessel maturation.

Angiogenesis is an essential process for correct development and physiology. This mechanism is tightly regulated by many signals that activate several pathways, which are constantly interacting with each other. There is mounting evidence that BMP9/ALK1 pathway is essential for a correct vessel maturation. Alterations in this pathway lead to the development of hereditary haemorrhagic telangiectasias. However, little was known about the BMP9 signalling cascade until the last years. Recent reports have shown that while BMP9 arrests cell cycle, it promotes the activation of anabolic pathways to enhance endothelial maturation. In light of this evidence, a new criterion for the classification of cytokines is proposed here, based on the physiological objective of the activation of anabolic routes. Whether this activation by a growth factor is needed to sustain mitosis or to promote a specific function such as matrix formation is a critical characteristic that needs to be considered to classify growth factors. Hence, the state-of-the-art of BMP9/ALK1 signalling is reviewed here, as well as its implications in normal and pathogenic angiogenesis.

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells.

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from 13C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.

Generation and Integrated Analysis of Advanced Patient-Derived Orthoxenograft Models (PDOX) for the Rational Assessment of Targeted Therapies in Endometrial Cancer.

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments.

Filamin B plays a key role in vascular endothelial growth factor-induced endothelial cell motility through its interaction with Rac-1 and Vav-2.

Actin-binding proteins filamin A (FLNA) and B (FLNB) are expressed in endothelial cells and play an essential role during vascular development. In order to investigate their role in adult endothelial cell function, we initially confirmed their expression pattern in different adult mouse tissues and cultured cell lines and found that FLNB expression is concentrated mainly in endothelial cells, whereas FLNA is more ubiquitously expressed. Functionally, small interfering RNA knockdown of endogenous FLNB in human umbilical vein endothelial cells inhibited vascular endothelial growth factor (VEGF)-induced in vitro angiogenesis by decreasing endothelial cell migration capacity, whereas FLNA ablation did not alter these parameters. Moreover, FLNB-depleted cells increased their substrate adhesion with more focal adhesions. The molecular mechanism underlying this effect implicates modulation of small GTP-binding protein Rac-1 localization and activity, with altered activation of its downstream effectors p21 protein Cdc42/Rac-activated kinase (PAK)-4/5/6 and its activating guanine nucleotide exchange factor Vav-2. Moreover, our results suggest the existence of a signaling complex, including FLNB, Rac-1, and Vav-2, under basal conditions that would further interact with VEGFR2 and integrin alphavbeta5 after VEGF stimulation. In conclusion, our results reveal a crucial role for FLNB in endothelial cell migration and in the angiogenic process in adult endothelial cells.

PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner.

BACKGROUND: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. METHODS: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. RESULTS: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. CONCLUSIONS: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.

FGFR Inhibition Overcomes Resistance to EGFR-targeted Therapy in Epithelial-like Cutaneous Carcinoma.

PURPOSE: Recurrent and/or metastatic unresectable cutaneous squamous cell carcinomas (cSCCs) are treated with chemotherapy or radiotherapy, but have poor clinical responses. A limited response (up to 45% of cases) to EGFR-targeted therapies was observed in clinical trials with patients with advanced and metastatic cSCC. Here, we analyze the molecular traits underlying the response to EGFR inhibitors, and the mechanisms responsible for cSCC resistance to EGFR-targeted therapy. EXPERIMENTAL DESIGN: We generated primary cell cultures and patient cSCC-derived xenografts (cSCC-PDXs) that recapitulate the histopathologic and molecular features of patient tumors. Response to gefitinib treatment was tested and gefitinib-resistant (GefR) cSCC-PDXs were developed. RNA sequence analysis was performed in matched untreated and GefR cSCC-PDXs to determine the mechanisms driving gefitinib resistance. RESULTS: cSCCs conserving epithelial traits exhibited strong activation of EGFR signaling, which promoted tumor cell proliferation, in contrast to mesenchymal-like cSCCs. Gefitinib treatment strongly blocked epithelial-like cSCC-PDX growth in the absence of EGFR and RAS mutations, whereas tumors carrying the E545K PIK3CA-activating mutation were resistant to treatment. A subset of initially responding tumors acquired resistance after long-term treatment, which was induced by the bypass from EGFR to FGFR signaling to allow tumor cell proliferation and survival upon gefitinib treatment. Pharmacologic inhibition of FGFR signaling overcame resistance to EGFR inhibitor, even in PIK3CA-mutated tumors. CONCLUSIONS: EGFR-targeted therapy may be appropriate for treating many epithelial-like cSCCs without PIK3CA-activating mutations. Combined EGFR- and FGFR-targeted therapy may be used to treat cSCCs that show intrinsic or acquired resistance to EGFR inhibitors.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Sunitinib inhibits tumor growth and synergizes with cisplatin in orthotopic models of cisplatin-sensitive and cisplatin-resistant human testicular germ cell tumors.

PURPOSE: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT. EXPERIMENTAL DESIGN: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed. RESULTS: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor alpha phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model. CONCLUSIONS: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.