Sawan Kumar Jha

CCBE1 Enhances Lymphangiogenesis via A Disintegrin and Metalloprotease With Thrombospondin Motifs-3–Mediated Vascular Endothelial Growth Factor-C Activation

Background —Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 ( CCBE1 ) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results —By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions —These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.Background— Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results— By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C. Conclusions— These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C–activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.

CCBE1 Enhances Lymphangiogenesis via ADAMTS3-Mediated VEGF-C Activation

Background —Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is a rare autosomal recessive disease, which is associated with mutations in the collagen- and calcium-binding EGF domains 1 ( CCBE1 ) gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for CCBE1 interactions with the VEGF-C growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results —By analyzing VEGF-C produced by CCBE1-transfected cells, we found that while CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 (ADAMTS3) protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector (AAV) mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by AAV-VEGF-C. Conclusions —These results identify ADAMTS3 as a VEGF-C activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.Background— Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results— By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C. Conclusions— These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C–activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.

Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells

BackgroundTriple negative breast cancer (TNBC) is a highly heterogeneous and aggressive type of cancer that lacks effective targeted therapy. Despite detailed molecular profiling, no targeted therapy has been established. Hence, with the aim of gaining deeper understanding of the functional differences of TNBC subtypes and how that may relate to potential novel therapeutic strategies, we studied comprehensive anticancer-agent responses among a panel of TNBC cell lines.MethodThe responses of 301 approved and investigational oncology compounds were measured in 16 TNBC cell lines applying a functional profiling approach. To go beyond the standard drug viability effect profiling, which has been used in most chemosensitivity studies, we utilized a multiplexed readout for both cell viability and cytotoxicity, allowing us to differentiate between cytostatic and cytotoxic responses.ResultsOur approach revealed that most single-agent anti-cancer compounds that showed activity for the viability readout had no or little cytotoxic effects. Major compound classes that exhibited this type of response included anti-mitotics, mTOR, CDK, and metabolic inhibitors, as well as many agents selectively inhibiting oncogene-activated pathways. However, within the broad viability-acting classes of compounds, there were often subsets of cell lines that responded by cell death, suggesting that these cells are particularly vulnerable to the tested substance. In those cases we could identify differential levels of protein markers associated with cytotoxic responses. For example, PAI-1, MAPK phosphatase and Notch-3 levels associated with cytotoxic responses to mitotic and proteasome inhibitors, suggesting that these might serve as markers of response also in clinical settings. Furthermore, the cytotoxicity readout highlighted selective synergistic and synthetic lethal drug combinations that were missed by the cell viability readouts. For instance, the MEK inhibitor trametinib synergized with PARP inhibitors. Similarly, combination of two non-cytotoxic compounds, the rapamycin analog everolimus and an ATP-competitive mTOR inhibitor dactolisib, showed synthetic lethality in several mTOR-addicted cell lines.ConclusionsTaken together, by studying the combination of cytotoxic and cytostatic drug responses, we identified a deeper spectrum of cellular responses both to single agents and combinations that may be highly relevant for identifying precision medicine approaches in TNBC as well as in other types of cancers.

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro

Cytosolic phospholipase A2 alpha (cPLA2α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA2α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA2α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA2α. Such promiscuous binding would explain the previous finding that cPLA2α has both PLA1 and PLA2 activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA2α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA2α.

Biology of Vascular Endothelial Growth Factor C in the Morphogenesis of Lymphatic Vessels

Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is vascular endothelial growth factor C (VEGF-C). VEGF-C is therefore an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most—if not all—applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate, e.g., to important aspects such as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.

Key molecules in lymphatic development, function, and identification

While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.

Abstract B21: Enhanced understanding of drug responses and drug-drug interactions in triple negative breast cancer cells with a multiplexed cell viability and cell death readout

Triple negative breast cancers (TNBC) are a heterogeneous group of cancers that remain a considerable clinical challenge with no known effective targeted therapy, limiting the therapies to cytotoxic chemotherapy, radiation and surgery. Molecular sub-classifications of TNBC have been established but none of these define therapy. With an aim to establish potential novel stratified therapeutic strategies to target TNBC, we applied the Drug Sensitivity and Resistance Testing (DSRT) (Pemovska et al., Cancer Discov. 2013) chemo-sensitivity profiling approach in which the responses of cells to 304 approved and investigational oncology drugs were explored. We studied a diverse panel of 15 TNBC cell lines in an attempt to functionally classify the TNBCs. To go beyond standard growth inhibition drug sensitivity testing we utilized a multiplexed cell viability and cytotoxicity readout allowing us to differentiate between cytostatic and cytotoxic drug responses. Evaluating both types of drug responses, several discoveries were made: First, the multiplexed viability and cytotoxicity readout identified several drug classes that previously had been assumed to have cytotoxic effects based on their strong effects on cell viability but in fact only showed cytostatic effects in most cells. Drug classes exhibiting this type of response included anti-mitotics, mTOR, CDK, metabolic inhibitors, as well as many targeted agents selectively inhibiting oncogenically activated pathways in individual cell lines. Second, within the responses to these broad cytostatic-acting classes of drugs, there were subsets of the cell lines that responded by cell death, suggesting that these cell lines may represent the TNBC subtypes that might best respond to the drugs in the clinic. To explore this further, we tested whether the cytostatic vs. cytotoxic responses could be linked to expression of protein biomarkers using a published TNBC cell line reverse-phase protein array data set (Daemen et al, Genome Biol. 2013). This led to the identification of potential predictive biomarkers such as PAI-1, MAPK phosphatase and Notch-3 levels linking to toxic responses to mitotic and proteasome inhibitors, suggesting that these could be explored as predictors for clinical responses for the drugs. Third, drug response based clustering of cell lines was very distinctive to transcriptomics based TNBC subgroups and recurrent mutation patterns, highlighting the considerable challenges in converting genetic and transcriptomic data to therapeutic responses. Fourth, the cytotoxicity readout highlighted effective synergistic drug combinations that were not apparent using cell viability readouts. For instance, we identified selective synergistic combinations of the MEK inhibitor trametinib with PARP inhibitors or the tyrosine kinase inhibitor ponatinib in DU4475 cells; as well as of the rapamycin analog everolimus with the ATP-competitive mTOR inhibitor dactolisib in several mTOR addicted cell lines. In conclusion, we showed that tracking drug-induced toxic responses in drug sensitivity testing and drug combination evaluation provides novel critical information on drug vulnerabilities. This argues that high throughput chemo-sensitivity profiling of cancer need to go beyond the current standard cell viability testing. Citation Format: Prson Gautam, Leena Karhinen, Agnieszka Szwajda, Sawan Kumar Jha, Bhagwan Yadav, Tero Aittokallio, Krister Wennerberg. Enhanced understanding of drug responses and drug-drug interactions in triple negative breast cancer cells with a multiplexed cell viability and cell death readout. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B21.

Abstract P6-02-01: Identification of subgroups of triple negative breast cancer cells with selective responses to mTOR, CDK, mitotic and proteasome inhibitors

Triple negative breast cancer (TNBC) is characterized by the lack of of estrogen, progesterone and HER2/ErbB2 receptors. It is a highly heterogeneous class of breast cancer and transcriptomics has recently been used to define 6 major subtypes of TNBC. We studied a panel of 15 TNBC cell lines using a chemical biology approach where we measure the responses to 306 approved and investigational oncology drugs. Clustering of cell lines based on their overall drug responses resulted in a strikingly different grouping compared to the gene expression derived one, highlighting that the current TNBC subtyping is not easily converted to differential sensitivities to drugs. To further evaluate the nature of the drug responses and to differentiate between their cell growth and cytotoxic effects, we multiplexed the standard cell viability readout in the cell line screening with detection of cytotoxicity. This simple multiplexed readout identified several drug classes that previously had been assumed to be cytotoxic based on strong effects on cell viability (cell numbers) while they in fact showed no or a very heterogeneous effect on cytotoxicity. Drug classes exhibiting this type of response included mTOR inhibitors, cyclin-dependent kinase inhibitors (eg. alvociclib), mitotic inhibitors (eg. paclitaxel) as well as proteasome inhibitors (eg. bortezomib) and RNA synthesis inhibitors (eg. dactinomycin). Further investigation of these drug classes showed that their static effects were reversible and in some cases the cells even overcame the inhibitory effect in the presence of the drug in a matter of a few days. Given the non-toxic responses to major classes of anticancer compounds such as mTOR inhibitors, we performed combination screens with these compounds to identify other drugs with which they may synergize to promote cancer cell specific killing. Surprisingly, we instead found that mTOR inhibitors had an antagonistic effect on the activity of many other cancer drugs such as different cytotoxic and antimitotic drugs, tyrosine kinase inhibitors, HDAC inhibitors and PARP inhibitors, suggesting that combining these classes of drugs may be counterproductive also in the clinic. We also found out that accessing a cytotoxic readout allowed us to identify effective synergistic drug combination concentrations that were not seen in cell viability readouts. For example, these synergistic toxic combination responses were seen in DU4475 cells when the MEK inhibitor trametinib was combined either with the PARP inhibitor iniparib or with the broad spectrum tyrosine kinase inhibitor ponatinib. In conclusion, multiplexed cell viability cell death readouts in drug sensitivity testing yields novel critical information on single drug and drug combination activities and liabilities. With this we were able to conclude that antimitotic, mTOR, CDK, proteasome and metabolic inhibitors have a heterogeneous cytotoxic effect across the panel of TNBC cell lines in contrast to their homogenous effect on metabolic inactivation. Citation Format: Prson Gautam, Leena Karhinen, Agnieszka Szwajda, Sawan Kumar Jha, Bhagwan Yadav, Tero Aittokallio, Krister Wennerberg. Identification of subgroups of triple negative breast cancer cells with selective responses to mTOR, CDK, mitotic and proteasome inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-01.