Verteporfin Treatment Research Articles

C-MYC and BCL-2 mediate YAP-regulated tumorigenesis in OSCC.

Transcriptional co-activator Yes-associated protein (YAP) is a key oncogene in mammalian cells. The present understanding of YAP in oral squamous cells carcinoma (OSCC) remains unclear. The purpose of this study is to investigate the effects of YAP on proliferation and apoptosis in OSCC and the molecular mechanism. The results showed the expression level of YAP was higher in OSCC tissues than that in adjacent normal tissues. Knockdown of YAP in CAL27 cell lines prohibited cell proliferation while augmented apoptosis. Conversely, overexpression of YAP protected cells from apoptosis and promoted cell proliferation. Moreover, C-MYC and BCL-2 mRNA and protein levels were altered due to the differential expression of YAP. Subsequent Verteporfin treatment in CAL27 cells revealed that the transcription and translation of BCL-2 and C-MYC both decreased. In a tumor xenograft model, knockdown of YAP suppressed tumor growth of CAL27 in vivo, while YAP overexpression promoted the tumor growth. These results suggest that YAP is a crucial regulator that exerts pro-proliferation and anti-apoptosis effects in OSCC through actions affecting the cell cycle and intrinsic apoptotic signaling. Thus YAP could potentially serve as a valuable molecular biomarker or therapeutic target in the treatment of OSCC.

In Vitro Validation of the Hippo Pathway as a Pharmacological Target for Canine Mammary Gland Tumors.

Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Some clinical and molecular similarities between certain CMT subtypes and breast cancer make them a potential model for the study of the human disease. As misregulated Hippo signaling is thought to play an important role in breast cancer development and also occurs in CMTs, we sought to determine if Hippo represents a valid pharmacological target for the treatment of CMTs. Six CMT cell lines were assessed for their expression of the Hippo pathway effectors YAP and TAZ and for their sensitivity to verteporfin, an inhibitor of YAP-mediated transcriptional coactivation. Four cell lines that expressed YAP (CMT-9, -12, -28, -47) were found to be very sensitive to verteporfin treatment, which killed the cells through induction of apoptosis with ED50 values of 14-79 nM. Conversely, two YAP-negative cell lines (CF-35, CMT-25) were an order of magnitude more resistant to verteporfin. Verteporfin suppressed the expression of YAP/TAZ target genes, particularly CYR61 and CTGF, which play important roles in breast cancer development. Verteporfin was also able to inhibit cell migration and anchorage-independent growth. Likewise, verteporfin efficiently suppressed tumor cell invasiveness in the CMT-28 and -47 lines, but not in CF-35 cells. Together, our findings provide proof of principle that pharmacological targeting of the Hippo pathway compromises the viability and attenuates the malignant behavior of CMT cells. These results will serve as the basis for the development of novel chemotherapeutic approaches for CMTs that could translate to human medicine.

Abstract 365: Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma

Abstract Hippo/YAP1 signaling pathway is a tumor suppressive pathway that controls the organ size by modulating the cell growth, proliferation and apoptosis and is conserved from Drosophila to mammals. In mammals, the Hippo tumor suppressor pathway consists of cascade of kinases in which MST1/2 phosphorylates and activates LATS1/2. The latter phosphorylates the oncogenic transcriptional coactivators YAP1 and TAZ, leading to their cytoplasmic retention by 14-3-3 proteins and/or degradation. Inactivation of MST and LATS kinases allows YAP1 and/ or TAZ nuclear translocation and subsequent activation of their target genes. Deregulation of Hippo pathway can induce tumors in model organisms and occurs in wide range of human cancers including melanoma. Merlin, a key component of this pathway which inhibits YAP1, is mutated/deleted in 8% of melanoma. Majority of uveal melanomas are driven by Gq/11 mutations that trigger YAP1 nuclear translocation, promoting tumor growth. High levels of YAP1 in BRAFV600E mutant tumors confer resistance to RAF- and MEK- targeted therapy in patients. Our results show that YAP1 level is elevated in melanoma and the YAP1 inhibitor verteporfin alone or in combination with B-RAF inhibitor PLX4720 reduces the viability, invasion and anchorage-independent growth of B-RAF V600E mutant SK-MEL-28 and SK-MEL-5 cells. In addition, verteporfin treatment also reduced the viability of PLX4720 resistant 1205 cells. Western blot analysis of verteporfin and PLX4720 treated SK-MEL-28 and SK-MEL-5 cells displayed reduced levels of YAP1, B-RAF, pERK, MEK and pMEK. We also report a novel physical interaction between YAP1 and B-RAF; this could be detected using double immunofluorescence and immunoprecipitation-western blotting techniques in both B-RAF V600E mutant and N-Ras mutant melanoma cells. Proximity ligation assays on tissue microarray showed that YAP1-B-RAF interaction is elevated in metastatic melanoma compared to normal skin. These novel findings highlight the crosstalk between B-RAF and Hippo/YAP1 signaling which might have a potential role in melanoma development and progression. Further, experiments are in progress to elucidate the functional significance of YAP1-B-RAF interaction in melanoma. Citation Format: Mohan Kumar Durai Raj, Jonathan Nguyen, Namrata Bora-singhal, Jane Messina, Geoffrey Gibney, Srikumar Chellappan. Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2017-365

Abstract 117: Anti-proliferative and cytotoxic effects of verteporfin in endometrial cancer cells

Abstract Endometrial cancer (EMCA) is the most prevalent gynecologic cancer in women in the United States. The relatively poor prognosis and limited therapeutic options in advanced endometrial cancer underscores the urgency in developing new and more effective treatment modalities. On the basis of clinical and histopathological features, EMCA is classified into type I and type II disease groups. Type 1 is estrogen potentiated, estrogen receptor (ER) and progesterone receptor (PR) positive, and generally carries a favorable prognosis. Type 2 is ER/PR negative and carries a much poorer prognosis. We investigated the therapeutic efficacy and mechanism of action of Verteporfin (VP), a benzoporphyrin derivative in EMCA. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. Similarly, VP treatment increased apoptosis in organoids, developed from patient-derived xenografts. Since the half-life of verteporfin is very short, we evaluated the longer duration efficacy of VP, encapsulated in mPEG-PLGA polymers. Our results demonstrated that VP nanoparticles (NP) decreased cell viability in both Type 1 (HEC-1-B) and Type 2 (ARK-1) EMCA cell lines over a period of 4 days. We did not observe any lethal effects of blank NP to the EMCA cells. In order to determine the efficacy of VP in changing cellular transcriptome, we performed RNASeq of EMCA cells after treatment with VP. RNASeq data analysis was carried out using latest version of Kallisto and Sleuth software. Based on the global gene expression whole transcriptome analysis, we observed that HEC-1-A VP treated cells show 225 differentially expressed transcripts (out of 204 genes); whereas HEC-1-B VP treated cells show 12315 differentially expressed transcripts (out of 7870 genes). There were 114 differentially expressed transcripts (out of 105 genes) common to both the cell lines. We observed that ATM, ATR, BRCA2, CDK4, ERBB2, JAK2, NF1, NOTCH and TET2 are some of the differentially expressed genes after VP treatment. To test the efficacy of VP under in vivo conditions, we developed a subcutaneous mouse model of EMCA using HEC-1-B-GFP cells. IP administration of VP in this model achieved 21% tumor regression. To more accurately simulate the results in vivo in a clinically relevant model, we have developed an orthotopic tumor model with IV injection of VP. Taken together, our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer. Citation Format: Radhika P. Gogoi, Jessica M. Castañeda-Gill, Raghu P. Metpally, Sarath B. Krishnamurthy, Jamboor K. Vishwanatha, David J. Carey, John D. Nash, Venkata Ramesh Dasari. Anti-proliferative and cytotoxic effects of verteporfin in endometrial cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2017-117

Abstract 5833: Targeting the transcription co-activator TAZ inhibits MYC-driven medulloblastoma

Abstract Medulloblastoma (MB), the most common malignant pediatric brain tumor, results in significant neurological, intellectual and physical disability or death. Four MB subtypes (WNT, SHH, Group 3 and 4) have recently been identified. Group 3 MB, the most aggressive subtype, is more frequently associated with gene amplification and/or protein overexpression of the MYC (c-Myc) oncogene, hereby referred to as MYC-driven MBs. It is still unclear how MB cells activate and maintain high MYC expression, as only a small portion of MYC-high MBs is caused by MYC genomic amplification. MYC-targeted MB therapies are also lacking. Targeting MYC upstream activators is a promising strategy for inhibiting MYC-driven MBs, when compared to direct MYC inhibition that is still challenging. Here, we focus on the transcription co-activator TAZ, a novel MYC upstream activator and a potential therapeutic target for inhibiting MYC-driven MBs. TAZ (transcriptional co-activator with PDZ-binding motif) is an oncogenic driver in multiple human cancers. TAZ and its paralog YAP form complexes with other transcription factors, such as TEAD, to activate downstream gene targets. TAZ and YAP are transcriptional effectors of various signaling pathways, such as the Hippo pathway. We found for the first time that TAZ is an essential MYC activator in MB cells. TAZ but not its paralog YAP is highly expressed in MYC-amplified MB cells and their xenografts. TAZ silencing downregulates MYC and inhibits MB cell proliferation. Enforced TAZ expression induces MYC expression in MB cells and promotes MB cell growth. We further showed that TAZ forms complex with transcription activator TEAD to bind to and transactivate MYC enhancers. For pharmacological TAZ targeting, we found that the FDA-approved drug Verteporfin (VP) inhibits TAZ-TEAD interaction, suppresses MYC expression and inhibits MB cell tumor propagation in vivo. We have also developed a nanoparticle formulation by using DSPE-mPEG2000 micelles to achieve VP delivery cross the blood-brain barrier in mice bearing brain tumor xenografts. In summary, our results provide novel insights into MYC upstream activators in MB, and also identify TAZ as a potential therapeutic target for inhibiting MYC-driven MBs. This knowledge may also be widely applicable to other TAZ/MYC-expressing human cancers, such as glioblastoma. Moreover, our studies support the effectiveness of VP treatment in pre-clinical MB models. These results and also the nanocarrier system for systemic VP delivery provide a solid foundation for VP clinical trials in patients with MB and likely other MYC-driven cancers. Note: This abstract was not presented at the meeting. Citation Format: Yingchao Xue, Mingyao Ying. Targeting the transcription co-activator TAZ inhibits MYC-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5833. doi:10.1158/1538-7445.AM2017-5833

Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway

Porphyrin derivatives, in particular verteporfin (VP), a photosensitizer initially designed for cancer therapy, have been identified as inhibitors of the YAP-TEAD interaction and transcriptional activity. Herein we report the efficient convergent synthesis of the dipyrrin half of protoporphyrin IX dimethyl ester (PPIX-DME), in which the sensitive vinyl group was created at the final stage by a dehydroiodination reaction. Two other dipyrrin derivatives were synthesized, including dipyrrin 19 [(Z)-2-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4-vinyl-1H-pyrrole], containing two vinyl groups. We found that VP and dipyrrin 19 showed significant inhibitory effects on TEAD transcriptional activity in MDA-MB-231 human breast cancer cells, whereas other compounds did not show significant changes. In addition, we observed a marked decrease in both YAP and TAZ levels following VP treatment, whereas dipyrrin 19 treatment primarily decreased the levels of YAP and receptor kinase AXL, a downstream target of YAP. Together, our data suggest that, due to their chemical structures, porphyrin- and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibit TEAD transcriptional activity.

Verteporfin exhibits YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells.

Endometrial Carcinoma (EMCA) is the most common gynecologic malignancy and the fourth most common malignancy in women in the United States. Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. Verteporfin (VP), a benzoporphyrin derivative, was identified as an inhibitor of YAP-TEAD interaction. We investigated the therapeutic efficacy and mechanism of VP in EMCA. The efficacy of VP on cell viability, cytotoxicity and invasion was assayed in EMCA cell lines. An organoid model system was also developed to test the effect of VP on apoptotic markers in an in vitro model system. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. These effects occurred as early as 15 minutes following treatment. Similarly, VP treatment versus vehicle control increased apoptosis in human organoid model systems. Quantitative RT-PCR, cDNA based RTPCR array analysis and western blotting were performed to investigate the mechanism of VP action. The cytotoxic and anti-proliferative effects appeared to be independent of its effect on YAP. Our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer.

Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Human Leukemia NB4 Cells without Light Activation.

Background and Aims: Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has recently been proven a suppressor of yes-associated protein (YAP) and has shown potential in anticancer treatment. However, its anti-human leukemia effects in NB4 cells remain unclear. In this study, we investigated the effects of VP on proliferation and apoptosis in human leukemia NB4 cells.Methods: NB4 cells were treated with VP for 24 h. The effects of VP on cell proliferation were determined using a Cell-Counting Kit-8 assay (CCK-8) assay and colony forming assay. Apoptosis and cell cycle were evaluated by flow cytometry (FCM). The protein levels were detected by western blot.Results: We found that VP inhibited the proliferation of NB4 cells in a concentration and time-dependent manner. FCM analysis showed that VP induced apoptosis in a concentration dependent manner and that VP treatment led to cell cycle arrest at G0/G1 phase. Moreover, VP significantly decreased the protein expression of YAP, p-YAP, Survivin, c-Myc, cyclinD1, p-ERK, and p-AKT. In addition, VP increased the protein expression of cleaved caspase3, cleaved PARP, Bax, and p-p38 MAPK.Conclusions: VP inhibited the proliferation and induced apoptosis in NB4 cells.

Verteporfin, a suppressor of YAP-TEAD complex, presents promising antitumor properties on ovarian cancer.

Yes-associated protein (YAP) is a key transcriptional coactivator of Hippo pathway and has been shown to be an oncoprotein in ovarian cancer (OC). Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has been recently proven to be a suppressor of YAP–TEAD complex and has shown potential in anticancer treatment. In this study, we aimed to explore the potential effect of VP in the treatment of OC. Our results showed that VP led to inhibition of proliferation in a time- and dose-dependent manner and to the suppression of migratory and invasive capacities of OC cells. Western blot and real-time polymerase chain reaction demonstrated that VP induced YAP cytoplasmic retention and deregulated inducible YAP and CCNs in OC cells. In vivo, VP exerted a significant effect on tumor growth in OVCAR8 xenograft mice, resulting in tumor nodules with lower average weight and reduced volume of gross ascites. In addition, VP treatment remarkably upregulated cytoplasmic YAP and phosphorylation YAP and downregulated CCN1 and CCN2, but exerted little effect on YAP-upstream components in Hippo pathway. In conclusion, our results suggested that VP may be a promising agent for OC, acting by suppressing YAP–TEAD complex.

Abstract 73: Yap Plays a Crucial Role in the Development of Cardiomyopathy in Lysosomal Storage Diseases

Rag proteins play a critical role in regulating lysosomal functions. Muscle-specific deletion of RagA and RagB (Rag A/B) results in lysosomal dysfunction, suppression of autophagy, and development of cardiomyopathy, reminiscent of lysosomal storage diseases (LSDs). We investigated the molecular mechanism by which cardiomyopathy is developed in RagA/B knockout (KO) mice. We generated cardiac-specific Rag A/BKO (RagA/BcKO) mice using αMHC-Cre. Similar to the muscle-specific KO mice, RagA/BcKO mice showed prominent cardiac hypertrophy (heart weight (HW)/ tibial length (TL): 12.35 ± 0.41 vs. 7.12 ± 0.15, p< 0.01) at 3 months of age. RagA/BcKO mice exhibited significantly greater phospho-histone H3-positive cardiomyocytes than control mice, suggesting that cardiomyocyte proliferation is stimulated in RagA/BcKO mice. However, RagA/BcKO mice exhibited a significantly smaller percentage of fractional shortening (%FS: 23.0 ± 0.82 vs. 45.8 ± 1.06, p< 0.01) and significantly greater mortality than control mice (50% vs. 0%, p<0.01). RagA/BcKO mouse hearts showed upregulation of β-MHC and α-smooth muscle actin, indicating de-differentiation. Yes-associated protein (YAP) is a transcription co-factor that controls growth and survival and is a key downstream target of the Hippo pathway. YAP associates with p62/SQSTM1 and is degraded through autophagy. YAP is significantly more accumulated (5.9-fold, p<0.05) in RagA/BcKO mouse hearts, where autophagy is inhibited and p62/SQSTM1 is accumulated, than in control hearts. In order to elucidate the role of YAP in mediating cardiomyopathy, we crossed RagA/BcKO mice with cardiac-specific heterozygous YAPKO mice. Heterozygous deletion of YAP in RagA/BcKO mice reduced the heart size (HW/ TL: 9.12 ± 0.10 vs. 11.9 ± 0.27, p< 0.05) and improved cardiac function (%FS: 35.0 ± 2.89 vs. 24.0 ± 0.58, p< 0.05) in RagA/BcKO mice. Pharmacological inhibition of YAP by verteporfin treatment also suppressed cardiac hypertrophy (HW/ TL: 8.92 ± 0.11 vs. 12.09 ± 0.30, p< 0.05) and heart failure (%FS: 33.3 ± 2.29 vs. 23.3 ± 0.63, p< 0.05) in RagA/BcKO mice. These results indicate that YAP plays a crucial role in the development of cardiomyopathy in RagA/BcKO mice, a mouse model of LSDs.

Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis.

The imbalance in cell proliferation and apoptosis is considered an important role in the pathogenesis of endometriosis, but the exact mechanisms remains unclear. A newly established signaling pathway–Hippo/Yes-associated protein (YAP) pathway plays a critical role in the proliferation and apoptosis processes. However, studies focusing on Hippo/YAP pathway and endometriosis are lacking. The objective was to explore the function of the Hippo/YAP pathway in endometriosis. The expression of YAP was first investigated in endometrium of women with or without endometriosis. The role of YAP in cell proliferation and apoptosis is identified by transfection of endometrial stromal cells (ESCs) in vitro, subsequent Verteporfin treatments in eutopic ESCs in vitro, and endometriosis animal model of nude mice in vivo. Our results revealed that increased expression of YAP and decreased expression of p-YAP in ectopic and eutopic endometrium compared with normal endometrium. YAP knockdown in eutopic ESCs decreased cell proliferation and enhanced cell apoptosis companied with decreased expression of TEAD1, CTGF, and B-cell lymphoma/leukemia (BCL)-2; whereas overexpression of YAP resulted in increased proliferation and decreased apoptosis of normal ESCs with increased expression of TEAD1, CTGF, and BCL-2. By chromatin immunoprecipitation qPCR CTGF and BCL-2 were identified as directly downstream target genes of YAP-TEAD1 active complex. Eutopic ESCs treated with Verteporfin revealed decreased proliferation and enhanced apoptosis whereas in endometriosis animal models of nude mice treated with Verteporfin, the size of endometriotic lesions was significantly reduced. Our study suggests that the Hippo/YAP-signaling pathway plays a critical role in the pathogenesis of endometriosis and should present a novel therapeutic method against endometriosis.

YAP/TAZ Are Mechanoregulators of TGF-β-Smad Signaling and Renal Fibrogenesis.

Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-β responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-β In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-β-induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-β stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-β to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-β/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-β signaling and renal fibrogenesis.

MNGO-08MENINGIOMA GROWTH INHIBITION AND RADIOSENSITIZATION BY THE SMALL MOLECULE YAP INHIBITOR VERTEPORFIN

Meningiomas frequently harbor inactivating mutations in the tumor suppressor protein Merlin (NF2), an upstream activator of the Hippo signaling pathway. This aberration drives tumor growth and aggressiveness through inactivation of this pathway and unchecked activation of its downstream effector, Yes-associated protein (YAP). The transcriptional coactivator YAP has been previously implicated in driving meningioma growth and malignant transformation of non-neoplastic arachnoid cells. YAP exerts its effect by direct binding to the TEAD family of transcription factors and transactivation of growth-related genes. However, the efficacy of pharmacologic modulation of this pathway in meningioma has yet to be explored. Verteporfin is a small molecule inhibitor of the porphyrin family, recently implicated as a direct inhibitor of YAP. Here, we report that Verteporfin modulates growth and radiation resistance in NF2 driven meningiomas through inhibition of the YAP-TEAD interaction. In particular, our data demonstrates that Verteporfin treatment in the NF2 mutant KT21MG1 human malignant meningioma cell line, results in a decreased activity of oncogenic YAP, resulting in decreased mRNA expression of YAP downstream targets (CTGF, Mcl-1 and ANKRD1) (p ≤ 0.05). Moreover, Verteporfin inhibits meningioma cell growth and proliferation in a dose dependent manner (MTT reduction and viable cell number) (p ≤ 0.05). Interestingly, pretreatment with Verteporfin results in increased sensitivity to irradiation in vitro (p ≤ 0.05). In addition, we explore the in vivo efficacy of Verteporfin treatment in a murine skull base xenograft model of human meningioma. Taken together, our results show that Verteporfin is a potent inhibitor of meningioma cell growth and radioresistance, making it an attractive drug candidate for treatment of this disease. Hence, this targeted therapy towards the NF2-YAP axis holds promising alternatives to current standard of care.

Abstract 4444: Small molecule drug Verteporfin inhibits TAZ/YAP-driven signaling and tumorigenicity of breast cancer cells

Abstract More than 1.6 million people worldwide are diagnosed with breast cancer (BC) each year, making it the second most common form of cancer. An essential step toward a safe and effective BC therapy is to develop clinically translatable strategies to target major oncogenic drivers in BC cells. Transcription co-activators TAZ and YAP, both of which are nuclear effectors of the Hippo pathway, have recently been identified as major oncogenic drivers in BC that promote tumor propagation, metastasis and resistance to current therapies. To date, pharmacological strategies for TAZ/YAP inhibition are still lacking. Here, we successfully inhibited TAZ/YAP-driven signaling in BC models by repurposing Verteporfin, an FDA-approved drug currently used to treat neovascular macular degeneration but not human cancers. We found that Verteporfin treatment inhibited the nuclear translocation and protein expression of TAZ/YAP as well as TAZ/YAP-driven signaling (e.g. EMT signaling) in MDA-MB-231 and BT-549 breast cancer cells. Verteporfin potently inhibited cell proliferation and induced apoptotic cell death up to 90% in BC cells. Systemic Verteporfin treatment was well tolerated in mice and inhibited the growth of orthotopic tumor xenografts derived from MDA-MB-231 cells by more than 100% (p<0.01). We further characterized the molecular response of BC cells to Verteporfin using RNA-seq followed by qPCR and western blot validation. We found that Verteporfin inhibited multiple EMT marker genes (e.g. Fibronectin 1, Vimentin, Snail and Slug). In addition, Verteporfin suppressed the expression of genes that mediate BC metastasis (e.g. ANGPTL4, COX2, EREG and MMP1), and also downregulated major components of signaling pathways (e.g. Notch and Integrin) that are essential for tumorigenicity of BC cells. Some of these genes had been identified as TAZ/YAP-activated gene targets, supporting the inhibition of TAZ/YAP-driving signaling by Verteporfin. A number of novel targets were also discovered. Furthermore, we found that transcription factor SOX4, a downstream target of TGFβ signaling, was activated in BC cells surviving from Verteporfin treatment. Manipulating SOX4 expression in BC cells changed Verteporfin sensitivity. Cells with enhanced SOX4 expression were more resistant to Verteporfin. These results elucidate a novel mechanism underlying Verteporfin resistance and suggest that Verteporfin and TGFβ inhibitors could work in combination to inhibit BC. In conclusion, we identified Verteporfin as a potential inhibitor of TAZ/YAP-driven signaling in BC, and established a global map of Verteporfin-response genes in BC cells. We systematically characterized the inhibitory effects of Verteporfin on BC cells at the molecular and cellular levels, and further identified SOX4-driven mechanism that mediates Verteporfin resistance. These results will facilitate the development of Verteporfin-based therapy for BC. Citation Format: Han Sun, Mingyao Ying. Small molecule drug Verteporfin inhibits TAZ/YAP-driven signaling and tumorigenicity of breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2015-4444

Evaluation of YAP as a therapeutic target in HCC and CCA

BackgroundFor Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. YAP, the effector of Hippo signaling pathway, is an oncoprotein that promotes cell proliferation and inhibits apoptosis. Genetic manipulation of YAP results in biliary hamartoma and HCC. Therefore, we hypothesize that YAP is driver of liver cancer and a potential therapeutic target.
MethodsExpression of YAP and its transcriptional targets GPC3 and Survivin was surveyed in normal human liver and primary liver cancer tissues. Efficacy of YAP inhibitor Vertepofin and Survivin inhibitor YM155 was tested in HCC and CCA cell lines with MTT assay.ResultsCompared to the non‐tumor tissue, nuclear YAP expression significantly increased in both HCC and ICC specimens. Nuclear YAP levels significantly correlate with nuclear Survivin levels in HCC and ICC tissues, but not with GPC3 in HCC tissues. We also found that verteporfin and YM155 suppressed HCC and CCA cell proliferation in vitro and had an additive effect to sorafenib treatment. Moreover, HCC cell lines with higher YAP expression were more sensitive to verteporfin treatment.ConclusionsOur findings suggested that YAP contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Small molecules that target YAP and Survivin activity could be a promising therapeutic strategy for treatment of HCC and CCA with high YAP expression.

The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation

Verteporfin (VP), a benzoporphyrin derivative, is clinically used in photodynamic therapy for neovascular macular degeneration. Recent studies indicate that VP may inhibit growth of hepatoma cells without photoactivation through inhibition of YAP-TEAD complex. In this study, we examined the effects of VP without light activation on human retinoblastoma cell lines. Verteporfin but not vehicle control inhibited the growth, proliferation and viability of human retinoblastoma cell lines (Y79 and WERI) in a dose-dependent manner and was associated with downregulation of YAP-TEAD associated downstream proto-oncogenes such as c-myc, Axl, and surviving. In addition VP affected signals involved in cell migration and angiogenesis such as CTGF, cyr61, and VEGF-A but was not associated with significant effect on the mTOR/autophagy pathway. Of interest the pluripotency marker Oct4 were downregulated by Verteporfin treatment. Our results indicate that the clinically used photosensitizer VP is a potent inhibitor of cell growth in retinoblastoma cells, disrupting YAP-TEAD signaling and pluripotential marker OCT4. This study highlights for the first time the role of the YAP-TEAD pathway in Retinoblastoma and suggests that VP may be a useful adjuvant therapeutic tool in treating Rb patients.

Recent trends in the management of maculopathy secondary to pathological myopia

Pathological myopia is a frequent cause of secondary visual disturbance in young individuals worldwide. Myopic maculopathy describes a spectrum of clinical changes that comprise the main cause of visual loss among highly myopic individuals. Our aim is to describe current trends in the medical and surgical management of maculopathy secondary to pathological myopia. The epidemiology, natural history, medical and surgical treatment modalities for choroidal neovascular membrane (CNV) and vitreomacular disorders secondary to pathological myopia (PM) are reviewed and evaluated. The medical and surgical treatment modalities in the management of myopic maculopathy have evolved over time. Laser photocoagulation, photodynamic therapy with verteporfin and other medical treatments have been superseded by the use of anti-vascular endothelial growth factor in the management of CNV secondary to PM. Surgical treatments are beneficial in the treatment of vitreomacular interface disorders such as macular hole retinal detachment and macular traction; however, primary success rates remain lower than those for non-myopic individuals. This updated clinical perspective demonstrates that CNV and vitreomacular disorders in pathological myopia are treatable conditions. There are numerous medical and surgical interventions that have significantly improved the outcome of myopic maculopathy and several others currently under investigation. Nonetheless, as technology advances, further well-designed studies are necessary to establish a uniform evidence-based approach for classification and treatment.

This issue of Acta, printed and electronic

The cover illustration shows a painting of Allvar Gullstrand painted in 1915, 4 years after he received the Nobel Prize for medicine. This issue is dedicated to the centennial of Allvar Gullstrand’s Nobel Prize. He is the only ophthalmologist ever to receive this prestigious price. Stefan Seregard writes an editorial commemorating this event. This is followed by an historical article by Ehinger and Grzybowski on the life and times of Allvar Gullstrand. Yilmaz et al. in USA, Spain and Ireland review the use of intravitreal bevacizumab for diabetic macular oedema. This is an extensive review of the literature for this new and important treatment modality. Lundström et al. in Sweden and Australia found the Catquest questionnaire useful for measuring self-assessed visual function before and after cataract surgery. Mönestam and Behndig found that light scattering and glistening in intraocular lenses does not affect visual acuity. Gazieva et al. in Denmark performed LASIK with two types of laser systems, both which effectively reduce high degrees of myopia with reasonable but slightly different predictability. Rijneveld et al. in the Netherlands found that emergency corneal grafts fail mostly due to host factors, not the quality of the donor tissue. Reinshagen et al. in Switzerland and Germany used mesenchymal stem cell in rabbits to treat total limbal stem cell deficiency. Heijl et al. in Sweden and USA report that intraocular pressure reduction with drug treatment depends on the baseline IOP level and suggest that the baseline IOP level should be documented when reporting treatment effect on intraocular pressure. Larsson et al. in Sweden used the Heidelberg retina tomography system to evaluate optic nerve heads in children. Lin et al. in Taiwan describe lacrimal canaliculitis in a series of patients. Mehlsen et al. in Denmark found that a calcium channel blocker and an angiotensin converting enzyme inhibitor did not influence retinal autoregulation in diabetic patients. Kook et al. in Germany found that intravitreal bevacizumab and/or triamcinolone do not provide long-term improvement for diabetic macular oedema. Kofoed et al. in Copenhagen found that hyperbaric oxygen exposure influenced electrophysiological components related to bipolar in Müller cells. It did not influence retinal thickness. Holton et al. in Denmark examined the influence of light source on contrast sensitivity in patients with AMD. They found that full spectral radiance incandescent light sources seem optimal. Available on http://www.actaophthalmologica.com Stalmans and a large international group review extensively the use of colour Doppler imaging in ocular blood flow research. Founti et al. in Greece and USA found good reproducibility between technicians in imaging retrobulbar blood flow in healthy volunteers. Frøen et al. in Norway and Hungary found that cells from the iris pigment epithelium have limited capabilities as retinal stem cells. Andjelić et al. in Slovenia found that lens capsule contraction with changing in calcium levels or mechanical stimulus. Sieving in USA explains Medline indexing and keywords. Bull in Bergen proposes that fireworks should be banned to reduce eye injuries. Basit et al. in Trondheim describe a case of Wernicke’s encephalopathy in a young boy. Russo et al. in Liverpool found scleral calcification in a case of recurrent melanoma after brachytherapy. Rama et al. in Italy report severe keratitis following corneal cross-linking for keratoconus. Jonas and Jonas in Germany compare peripapillary scleral thickness in glaucoma patients and normal subjects. Lim and An in Korea report good results after epiretinal membrane removal. Kernt et al. in Germany had good experiences with focal and panretinal photocoagulation with a navigated laser. Fielden et al. in Canada describe a cluster of cases with intraocular inflammation following bevacizumab injection. Bloch et al. in Denmark and Egypt describe neovascular membrane contraction and retinal pigment epithelial rupture following verteporfin and bevacizumab treatment. Caramoy et al. in Germany found recurrent choroidal neovascular membrane after autologous RPE transplantation. I wish all readers a Merry Christmas and a Happy New Year and hope that they enjoy reading ACTA during the holidays.

Contraction of occult choroidal neovascular membrane and rupture of the retinal pigment following verteporfin and bevacizumab treatment

Contraction of occult choroidal neovascular membrane and rupture of the retinal pigment following verteporfin and bevacizumab treatment

Effects of combination therapy with verteporfin photodynamic therapy and ranibizumab in patients with age-related macular degeneration

This open-label, prospective, small-scale study investigated the benefits of same-day verteporfin and intravitreal ranibizumab in patients with predominantly classic, minimally classic or occult subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration. Patients received verteporfin at baseline and at month 3, if leakage persisted. Ranibizumab (0.5 mg) was given at baseline and months 1, 2 and 3, and thereafter at monthly intervals if required. Same-day ranibizumab was given ≥ 1 hr after verteporfin. Fifteen patients [11 male, four female; mean age 75.5 years (range 54-94 years)] were treated. At day 360, mean visual acuity (VA) had improved by 10.9 letters. An increase of ≥ 15 and ≥ 30 letters (i.e. ≥ 3 and ≥ 6 lines) was observed in seven (47%) patients and one patient (7%), respectively. Mean central retinal thickness (CRT) decreased by 85 μm. At days 7, 14 and 30, CNV perfusion was absent in 14/15 patients. Mean lesion area had reduced from baseline by 23.1% at day 120, 25.5% at day 180 and 23.6% at day 360. There were no visual safety concerns and intraocular pressures remained normal. Only two serious adverse events were recorded over the 12-month period, and neither was considered to be related to treatment. Same-day verteporfin plus ranibizumab improved VA, reduced CRT, prevented CNV perfusion and reduced lesion area safely over 12 months. Further investigation is warranted to confirm whether this combination improves long-term vision and reduces the need for retreatment.