It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Doxycycline-Induced Changes in Circulating MMP or TIMP2 Levels Are Not Associated with Skeletal-Related Event-Free or Overall Survival in Patients with Bone Metastases from Breast Cancer.

Doxycycline is often used as a promoter of inducible gene expression in preclinical models; however, it can also have direct effects on tumor growth and survival. This is due in part to its ability to inhibit cell invasion and regulate matrix metalloproteinase (MMP) expression. Given that doxycycline is also osteotropic, a clinical study to assess its effects on modulation of tumor progression or prevention of skeletal-related events (SRE) in patients with bone metastases from breast cancer (the Achilles trial) was undertaken. Patients received 100 mg of oral doxycycline twice daily for 12 weeks, with serum obtained at baseline and 4, 8 and 12 weeks post-initiation of doxycycline treatment. Exploratory analysis of the effects of doxycycline on circulating levels of MMP or tissue inhibitor of matrix metalloproteinase 2 (TIMP2) was performed in enrolled patients. Statistically significant associations were observed between MMP2, MMP9 and TIMP2 at baseline with significant associations maintained between absolute levels and changes in levels of MMP2 and TIMP2 at weeks 4-12 post initiation of doxycycline. Treatment with doxycycline generally resulted in decreases in MMP2 and MMP9 levels with concurrent upregulation of TIMP2 at 12 weeks post-initiation of doxycycline treatment. Despite this, we observed no association with the levels of any of these factors with either SRE-free or overall survival in this patient cohort. In summary, despite observing hypothesized effects of doxycycline administration on surrogate markers of its anti-tumor activity, measures of circulating levels of these biomarkers were not prognostic in this patient population.

Focal adhesion kinase inhibitors prevent osteoblast mineralization in part due to suppression of Akt-mediated stabilization of osterix.

Focal Adhesion Kinase (FAK) is an important regulator of tumor cell proliferation, survival and metastasis. As such it has become a therapeutic target of interest in cancer. Previous studies suggested that use of FAK tyrosine kinase inhibitors (TKIs) blocks osteolysis in in vivo models of bone metastasis. However, from these studies it was not clear whether FAK TKIs blocked bone degradation by osteoclasts or also promoted bone formation by osteoblasts. In this study we evaluated whether use of the FAK TKI PF-562,271 affected the differentiation of pre-osteoblasts, or activity of mature differentiated osteoblasts. MC3T3-E1 pre-osteoblastic cells were treated with various doses of PF-562,271 following 3 or 10 days of differentiation which led to the inhibition of alkaline phosphatase (ALP) expression and reduced viable cell numbers in a dose-dependent manner. MC3T3-E1 cells which had been differentiated for 21 days prior to treatment with PF-562,271 showed a dose dependent decrease in mineralization as assessed by Alizarin Red staining, with concomitant decreased expression of ALP which is known to facilitate the bone mineralization activity of osteoblasts, however mRNA levels of the transcription factors RUNX2 and osterix which are important for osteoblast maturation and mineralization appeared unaffected at this time point. We speculated that this may be due to altered function of RUNX2 protein due to inhibitory phosphorylation by GSK3ß. We found treatment with PF-562,271 resulted in increased GSK3ß activity as measured by reduced levels of phospho-Ser9-GSK3ß which would result in phosphorylation and inhibition of RUNX2. Treatment of 21 day differentiated MC3T3-E1 cells with PF-562,271 in combination with GSK3ß inhibitors partially restored mineralization however this was not statistically significant. As we observed that FAK TKI also resulted in suppression of Akt, which is known to alter osterix protein stability downstream of RUNX2, we examined protein levels by western blot and found a dose-dependent decrease in osterix in FAK TKI treated differentiated MC3T3-E1 cells which is likely responsible for the reduced mineralization observed. Taken together our results suggest that use of FAK TKIs as therapeutics in the bone metastatic setting may block new bone formation as an off-target effect and thereby exacerbate the defective bone regulation that is characteristic of the bone metastatic environment.

VIVA1: a more invasive subclone of MDA-MB-134VI invasive lobular carcinoma cells with increased metastatic potential in xenograft models.

BACKGROUND: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. As few tools exist to study ILC metastasis, we isolated ILC cells with increased invasive properties to establish a spontaneously metastasising xenograft model. METHODS: MDA-MB-134VI ILC cells were placed in transwells for 7 days. Migrated cells were isolated and expanded to create the VIVA1 cell line. VIVA1 cells were compared to parental MDA-MB-134VI cells in vitro for ILC marker expression and relative proliferative and invasive ability. An intraductally injected orthotopic xenograft model was used to assess primary and metastatic tumour growth in vivo. RESULTS: Similar to MDA-MB-134VI, VIVA1 cells retained expression of oestrogen receptor (ER) and lacked expression of E-cadherin, however showed increased invasion in vitro. Following intraductal injection, VIVA1 and MDA-MB-134VI cells had similar primary tumour growth and survival kinetics. However, macrometastases were apparent in 7/10 VIVA1-injected animals. Cells from a primary orthotopic tumour (VIVA-LIG43) were isolated and showed similar proliferative rates but were also more invasive than parental cells. Upon re-injection intraductally, VIVA-LIG43 cells had more rapid tumour growth with similar metastatic incidence and location. CONCLUSIONS: We generated a new orthotopic spontaneously metastasising xenograft model for ER+ ILC amenable for the study of ILC metastasis.

Mapping vitamin B6 metabolism by hydrazoCEST magnetic resonance imaging.

A new CEST-MRI contrast agent, 2-HYNIC, capable of sensing aromatic aldehydes is reported. Pyridoxal 5'-phosphate, a key Vitamin B6 metabolite necessary for >140 biotransformations was mapped by CEST-MRI in vitro and in vivo in lung cancer. 2-HYNIC provided access to this key biomarker associated with a variety of human diseases.

Targeting Intercellular Communication in the Bone Microenvironment to Prevent Disseminated Tumor Cell Escape from Dormancy and Bone Metastatic Tumor Growth.

Metastasis to the bone is a common feature of many cancers including those of the breast, prostate, lung, thyroid and kidney. Once tumors metastasize to the bone, they are essentially incurable. Bone metastasis is a complex process involving not only intravasation of tumor cells from the primary tumor into circulation, but extravasation from circulation into the bone where they meet an environment that is generally suppressive of their growth. The bone microenvironment can inhibit the growth of disseminated tumor cells (DTC) by inducing dormancy of the DTC directly and later on following formation of a micrometastatic tumour mass by inhibiting metastatic processes including angiogenesis, bone remodeling and immunosuppressive cell functions. In this review we will highlight some of the mechanisms mediating DTC dormancy and the complex relationships which occur between tumor cells and bone resident cells in the bone metastatic microenvironment. These inter-cellular interactions may be important targets to consider for development of novel effective therapies for the prevention or treatment of bone metastases.

Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells.

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that, in turn, promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report we analyzed clinical datasets of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We, thus, developed a new therapeutic approach to inhibit EGFR and hypoxia by combination treatment with metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24- and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic; this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.

The plasma peptides of breast versus ovarian cancer.

BACKGROUND: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC-ESI-MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. CONCLUSION: There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC-ESI-MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

The plasma peptidome.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC-ESI-MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. METHODS: A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC-ESI-MS/MS experiments analyzed in SQL Server R. RESULTS: Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 µL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey-Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. CONCLUSIONS: The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC-ESI-MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

Co-inhibition of mTORC1, HDAC and ESR1α retards the growth of triple-negative breast cancer and suppresses cancer stem cells.

Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer. It causes the majority of breast cancer-related deaths, which has been largely associated with the plasticity of tumor cells and persistence of cancer stem cells (CSCs). Conventional chemotherapeutics enrich CSCs and lead to drug resistance and disease relapse. Development of a strategy capable of inhibiting both bulk and CSC populations is an unmet medical need. Inhibitors against estrogen receptor 1, HDACs, or mTOR have been studied in the treatment of TNBC; however, the results are inconsistent. In this work, we found that patient TNBC samples expressed high levels of mTORC1 and HDAC genes in comparison to luminal breast cancer samples. Furthermore, co-inhibition of mTORC1 and HDAC with rapamycin and valproic acid, but neither alone, reproducibly promoted ESR1 expression in TNBC cells. In combination with tamoxifen (inhibiting ESR1), both S6RP phosphorylation and rapamycin-induced 4E-BP1 upregulation in TNBC bulk cells was inhibited. We further showed that fractionated CSCs expressed higher levels of mTORC1 and HDAC than non-CSCs. As a result, co-inhibition of mTORC1, HDAC, and ESR1 was capable of reducing both bulk and CSC subpopulations as well as the conversion of fractionated non-CSC to CSCs in TNBC cells. These observations were partially recapitulated with the cultured tumor fragments from TNBC patients. Furthermore, co-administration of rapamycin, valproic acid, and tamoxifen retarded tumor growth and reduced CD44high/+/CD24low/- CSCs in a human TNBC xenograft model and hampered tumorigenesis after secondary transplantation. Since the drugs tested are commonly used in clinic, this study provides a new therapeutic strategy and a strong rationale for clinical evaluation of these combinations for the treatment of patients with TNBC.

Dual inhibition of Wnt and Yes-associated protein signaling retards the growth of triple-negative breast cancer in both mesenchymal and epithelial states.

Triple-negative breast cancer (TNBC), the most refractory subtype of breast cancer to current treatments, accounts disproportionately for the majority of breast cancer-related deaths. This is largely due to cancer plasticity and the development of cancer stem cells (CSCs). Recently, distinct yet interconvertible mesenchymal-like and epithelial-like states have been revealed in breast CSCs. Thus, strategies capable of simultaneously inhibiting bulk and CSC populations in both mesenchymal and epithelial states have yet to be developed. Wnt/ß-catenin and Hippo/YAP pathways are crucial in tumorigenesis, but importantly also possess tumor suppressor functions in certain contexts. One possibility is that TNBC cells in epithelial or mesenchymal state may differently affect Wnt/ß-catenin and Hippo/YAP signaling and CSC phenotypes. In this report, we found that YAP signaling and CD44high /CD24-/low CSCs were upregulated while Wnt/ß-catenin signaling and ALDH+ CSCs were downregulated in mesenchymal-like TNBC cells, and vice versa in their epithelial-like counterparts. Dual knockdown of YAP and Wnt/ß-catenin, but neither alone, was required for effective suppression of both CD44high /CD24-/low and ALDH+ CSC populations in mesenchymal and epithelial TNBC cells. These observations were confirmed with cultured tumor fragments prepared from patients with TNBC after treatment with Wnt inhibitor ICG-001 and YAP inhibitor simvastatin. In addition, a clinical database showed that decreased gene expression of Wnt and YAP was positively correlated with decreased ALDH and CD44 expression in patients' samples while increased patient survival. Furthermore, tumor growth of TNBC cells in either epithelial or mesenchymal state was retarded, and both CD44high /CD24-/low and ALDH+ CSC subpopulations were diminished in a human xenograft model after dual administration of ICG-001 and simvastatin. Tumorigenicity was also hampered after secondary transplantation. These data suggest a new therapeutic strategy for TNBC via dual Wnt and YAP inhibition.

The plasma peptides of ovarian cancer.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. METHODS: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 µl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test. CONCLUSION: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2.

The importance of microRNA (miRNA) to vascular biology is becoming increasingly evident; however, the function of a significant number of miRNA remains to be determined. In particular, the effect of growth factor regulation of miRNAs on endothelial cell morphogenesis is incomplete. Thus, we aimed to identify miRNAs regulated by pro-angiogenic vascular endothelial growth factor (VEGF) and determine the effects of VEGF-regulated miRNAs and their targets on processes important for angiogenesis. Human umbilical vein endothelial cells (HUVECs) were thus stimulated with VEGF and miRNA levels assessed using microarrays. We found that VEGF altered expression of many miRNA, and for this study focused on one of the most significantly down-regulated miRNA in HUVECs following VEGF treatment, miR-30b. Using specific miRNA mimics, we found that overexpression of miR-30b inhibited capillary morphogenesis in vitro, while depletion of endogenous miR-30b resulted in increased capillary morphogenesis indicating the potential significance of down-regulation of miR-30b as a pro-angiogenic response to VEGF stimulation. MiR-30b overexpression in HUVEC regulated transforming growth factor beta 2 (TGFß2) production, which led to increased phosphorylation of Smad2, indicating activation of an autocrine TGFß signaling pathway. Up-regulation of TGFß2 by miR-30b overexpression was found to be dependent on ATF2 activation, a transcription factor known to regulate TGFß2 expression, as miR-30b overexpressing cells exhibited increased levels of phosphorylated ATF2 and depletion of ATF2 inhibited miR-30b-induced TGFß2 expression. However, miR-30b effects on ATF2 were indirect and found to be via targeting of the known ATF2 repressor protein JDP2 whose mRNA levels were indirectly correlated with miR-30b levels. Increased secretion of TGFß2 from HUVEC was shown to mediate the inhibitory effects of miR-30b on capillary morphogenesis as treatment with a neutralizing antibody to TGFß2 restored capillary morphogenesis to normal levels in miR-30b overexpressing cells. These results support that the regulation of miR-30b by VEGF in HUVEC is important for capillary morphogenesis, as increased miR-30b expression inhibits capillary morphogenesis through enhanced expression of TGFß2.

A phase II trial of dovitinib in previously-treated advanced pleural mesothelioma: The Ontario Clinical Oncology Group.

OBJECTIVES: Following failure of a platinum-antifolate combination regimen, there is no standard therapy for advanced malignant pleural mesothelioma (MPM). The fibroblast growth factor receptor (FGFR) signaling pathways may be a relevant target in MPM. Dovitinib inhibits multiple tyrosine receptor kinases, predominantly the vascular endothelial growth factor receptors (VEGFR), but also FGFRs, and could be active in MPM. METHODS: This open-label multicentre phase II trial [NCT01769547] enrolled fit, consenting adult patients with advanced MPM who had previously received platinum-antifolate combination chemotherapy and up to one additional line of systemic therapy. Dovitinib was administered orally at 500mg/day for 5days on, 2days off, in 28-day cycles. Response was assessed every 2 cycles using RECIST 1.1 criteria modified for MPM. Correlative studies included FGFR-1 amplification on archival tumour and serum samples for circulating angiogenesis factors. The primary end-point was the proportion of patients progression-free at 3 months (PF3) using a two-stage design. RESULTS: 12 patients (10 males, median age 67) were enrolled. The median number of cycles administered was 2.5 (range 1-8). One unconfirmed partial response was observed. PF3 was 50% (95% confidence interval 28.4% to 88.0%); although the criterion for proceeding to stage II accrual was met, the trial was halted due to a combination of minimal activity with several early progression events and poor tolerability in this patient population. One of 12 tumour specimens had low amplification of FGFR-1. CONCLUSIONS: Dovitinib has minimal activity in previously-treated MPM. The role of the FGFR pathway in MPM remains unclear.

Strategies for obtaining bone biopsy specimens from breast cancer patients - Past experience and future directions.

BACKGROUND: Cancer and its treatment can have multiple effects on the bone. Despite the widespread use of in vivo and in vitro models, it is still necessary to understand these effects in humans. Obtaining human bone biopsies is technically challenging and in this article we review the experiences from the Ottawa Bone Oncology Program. METHODS: A series of bone biopsy studies in breast cancer patients with and without bone metastasis have been performed. We reviewed the results of these studies and present them in a descriptive manner. We discuss lessons learned from each project and how they have affected future directions for research. RESULTS: Since 2009, 5 studies have been performed accruing 97 breast cancer patients. Study endpoints have ranged from comparing the yield of malignant cells from CT-guided versus standard iliac crest biopsies, to studies assessing the feasibility of micro-CT analysis on Jedhadi trephines to evaluate bisphosphonate effects on bone micro-architecture. More recently, we have assessed the feasibility of performing repeat bone biopsies in the same patient as well as evaluating the practicality of obtaining bone tissue at the time of orthopaedic surgery. CONCLUSION: Human bone tissue is an important biological resource. Our experience suggests that obtaining bone biopsies is feasible and can yield adequate amount of tumour cells for many studies. However, these remain technically challenging specimens to obtain and given the rapid advances in cancer therapeutics and the use of potent adjuvant bone-targeted agents, more centres need to be involved in these types of studies.

Both bulk and cancer stem cell subpopulations in triple-negative breast cancer are susceptible to Wnt, HDAC, and ERα coinhibition.

Development of targeted therapies for triple-negative breast cancer (TNBC, a more aggressive subtype) is an unmet medical need. We analyzed data from 887 patients with invasive breast cancer and observed that increased Wnt and histone deacetylase (HDAC) activities are associated with estrogen receptor 1 (ESR1) and progesterone receptor (PGR) repression, poor survival, and increased relapse. The inverse correlation between Wnt signaling and repression of ESR1 and PGR expression was found to be magnified in cancer stem cell (CSC) subpopulations in TNBC cell lines. Cosuppression of Wnt, HDAC, and ESR1 using clinically relevant low-dose inhibitors effectively repressed both bulk and CSC subpopulations and converted CSCs to non-CSCs in TNBC cells without affecting MCF-10A mammary epithelial cells.

Future directions for bone metastasis research - highlights from the 2015 bone and the Oncologist new updates conference (BONUS).

In an era of reduced peer-reviewed grant funding, performing academic bone oncology-related research has become increasingly challenging. Over the last 10 years we have held an annual meeting to bring together clinicians, clinician/scientists and basic biomedical researchers interested in the effects of cancer and its treatment on skeletal tissues. In the past these "Bone and the Oncologist New Updates Conference (BONUS)" meetings have served as critical catalyst for initiating productive research collaborations between attendees. The 2015 BONUS meeting format focused on potential key research themes that could form the basis of a coordinated national research strategy to tackle unmet clinical and research needs related to complications associated with cancer metastasis to bone. The three themes planned for discussion were: Is bone metastases-related pain the main issue facing patients? Are there new therapeutic targets for patients with bone metastases? How do we more firmly link basic science with clinical practice? We present a summary of lectures and commentaries from the attendees to serve as an example that other similarly motivated groups can model and share their experiences. It is our hope that these presentations will result in comments, feedback and suggestions from all those researchers interested in this important area.

Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer.

Blockade of epidermal growth factor receptor (EGFR) activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC). As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs), there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK) activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975) were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271) both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be particularly sensitive to this combination treatment. As such, further evaluation of this combination therapy is warranted and could prove to be an effective therapeutic approach for patients with inherent EGFR TKI-resistant NSCLC.

Issues Affecting the Loco-regional and Systemic Management of Patients with Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) of the breast is the second most common type of invasive breast carcinoma accounting for 8-14% of all breast cancers. Traditional management of ILC has followed similar paradigms as that for invasive ductal carcinoma (IDC). However, ILC represents a pathologically, clinically and biologically unique variant of breast cancer with particular management challenges. These challenges are seen in both the loco-regional management of ILC; where ILC tumors tend to avoid detection and hence present as more clinically advanced and surgically challenging carcinomas, and the systemic management with a unique response pattern to standard systemic therapies. Because of these challenges, the outcome for patients with ILC has likely lagged behind the continued improvements seen in outcome for patients with IDC. Here, we discuss some of the unique challenges ILC presents and discuss possible management strategies to best overcome the difficulties in the loco-regional and systemic management of patients with ILC.

Cardamonin reduces chemotherapy-enriched breast cancer stem-like cells in vitro and in vivo.

The failure of cytotoxic chemotherapy in breast cancers has been closely associated with the presence of drug resistant cancer stem cells (CSCs). Thus, screening for small molecules that selectively inhibit growth of CSCs may offer great promise for cancer control, particularly in combination with chemotherapy. In this report, we provide the first demonstration that cardamonin, a small molecule, selectively inhibits breast CSCs that have been enriched by chemotherapeutic drugs. In addition, cardamonin also sufficiently prevents the enrichment of CSCs when simultaneously used with chemotherapeutic drugs. Specifically, cardamonin effectively abolishes chemotherapeutic drug-induced up-regulation of IL-6, IL-8 and MCP-1 and activation of NF-κB/IKBα and Stat3. Furthermore, in a xenograft mouse model, co-administration of cardamonin and the chemotherapeutic drug doxorubicin significantly retards tumor growth and simultaneously decreases CSC pools in vivo. Since cardamonin has been found in some herbs, this work suggests a potential new approach for the effective treatment of breast CSCs by administration of cardamonin either concurrent with or after chemotherapeutic drugs.