Ghada M. Sharif

Impact of Fibroblast Growth Factor-Binding Protein–1 Expression on Angiogenesis and Wound Healing

Fibroblast growth factors (FGFs) participate in embryonic development, in maintenance of tissue homeostasis in the adult, and in various diseases. FGF-binding proteins (FGFBP) are secreted proteins that chaperone FGFs stored in the extracellular matrix to their receptor, and can thus modulate FGF signaling. FGFBP1 (alias BP1, FGF-BP1, or HBp17) expression is required for embryonic survival, can modulate FGF-dependent vascular permeability in embryos, and is an angiogenic switch in human cancers. To determine the function of BP1 in vivo, we generated tetracycline-regulated conditional BP1 transgenic mice. BP1-expressing adult mice are viable, fertile, and phenotypically indistinguishable from their littermates. Induction of BP1 expression increased mouse primary fibroblast motility in vitro, increased angiogenic sprouting into subcutaneous matrigel plugs in animals and accelerated the healing of excisional skin wounds. FGF-receptor kinase inhibitors blocked these effects. Healing skin wounds showed increased macrophage invasion as well as cell proliferation after BP1 expression. Also, BP1 expression increased angiogenesis during the healing of skin wounds as well as after ischemic injury to hindlimb skeletal muscles. We conclude that BP1 can enhance FGF effects that are required for the healing and repair of injured tissues in adult animals.

Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

The nuclear coactivator amplified in breast cancer 1 maintains tumor-initiating cells during development of ductal carcinoma in situ

The key molecular events required for the formation of ductal carcinoma in situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here, we show that the nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal three-dimensional mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both before DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24−/CD44+ breast cancer-initiating cell (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. The loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, human epidermal growth factor receptor 2 (HER2) and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 has a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through the maintenance of BCIC, facilitates formation of DCIS, a necessary step before development of invasive disease.

Cell density regulates cancer metastasis via the Hippo pathway

Metastatic spread of cancer cells from the primary tumor site to distant organs is the major cause of death in cancer patients. To disseminate, cancer cells detach from the primary tumor, enter the blood stream and extravasate at distant organ sites such as the liver, lung, bone or brain. While cancer cells are known to evade contact inhibition during growth in culture, we found that cell density is still sensed and can signal through the Hippo pathway effectors LATS1 and YAP. These effectors control cancer cell invasive behavior into stromal tissues, expression of cytokines that recruit inflammatory cells and progression toward metastatic spread. In this perspective, we discuss the drivers and the significance of pathways controlled by cell growth density.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish ( Danio Rerio )

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Abstract 2357: CDK4/6 inhibition in early stage triple negative breast cancer

Palbociclib (PD), a CDK4/6 inhibitor, has recently been approved for use in advanced ER positive breast cancer, demonstrating significant benefit in combination with endocrine therapies. Ductal carcinoma in situ (DCIS) is an inobligate precursor to invasive ductal carcinoma with anywhere from 14-53% of cases progressing. Progression is unpredictable and driven by poorly understood mechanisms. In this study, we assess the efficacy of PD in the context of this early stage disease using a triple negative DCIS model(MCF10DCIS), not only in inhibiting cell cycle but also in hindering the progression from in situ to invasive lesion. We demonstrate similar efficacy of PD in 2D on MCF10DCIS and immortal mammary epithelial cells (MCF10As) in preventing proliferation, inhibiting phosphorylation of RB and stopping cell cycle progression. Yet in 3D, MCF10DCIS cells form smaller, more organized spheres in the presence of drug while MCF10As appear unaffected, highlighting potential disease selectivity in vivo. Interestingly, when a cDNA array is performed on MCF10DCIS cells grown in 3D +/- PD, the most regulated genes are not well characterized cell cycle genes as one might predict based on previously published data and 2D response to treatment, suggesting alternative CDK4/6 targets may be responsible for the phenotype observed in 3D. We also demonstrate in a nude mouse xenograft model of MCF10DCIS that PD is able to significantly reduce overall tumor burden while simultaneously delaying the progression of lesions to invasive disease. Though the growth of PD treated MCF10DCIS lesions is delayed we demonstrate that proliferation is unaffected in vivo by the presence of the drug, based on equivalent Ki67 signal within the lesions. This reiterates the possibility that in 3D and in vivo contexts PD targets mechanisms outside of cell cycle regulation in order to slow tumor growth and delay invasive transition. We believe that investigation into alternative targets of CDK4/6 altered by PD in 3D and in vivo may provide insights on important mechanisms of DCIS progression in DCIS models and human patients. Citation Format: William B. Kietzman, Virginie Ory, Fransisco Saenz, Ghada Sharif, Anton Wellstein, Anna T. Riegel. CDK4/6 inhibition in early stage triple negative breast cancer [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 2357. doi:10.1158/1538-7445.AM2017-2357

Abstract A15: SMC2 role in regulating tumor angiogenesis via FGF signaling

Fibroblast growth factors (FGFs) play a crucial role during embryonic development, tissue homeostasis, wound healing and angiogenesis. FGF-binding protein 1 (FGFBP1) is a secreted protein that binds FGFs stored in the extracellular matrix and transports them to their receptor, therefore they can modulate FGF signaling. FGFBP1 contributes to the progression of many cancers including colon, pancreas and squamous cell cancer. It acts as an angiogenic switch during tumor development. E0771 cells, a murine mammary carcinoma cell line, was subcutaneously injected into syngeneic C57BL/6 mice. While these cells form aggressive tumors within two weeks and metastasize to the lungs in wild type FGFBP1 (FGFBP1+/+) mice, there was no tumor growth or metastasis in FGFBP1 knock out (FGFBP1-/-) mice. A genome-wide shRNA library screen revealed a novel role for structural maintenance of the chromosome 2 (SMC2) in compensating for the loss of FGFBP1 in the stroma of FGFBP1-/- mice. E0771 cells with SMC2 knock down (KD) or CRISPR targeting SMC2 formed aggressive tumors in FGFBP1-/- mice and metastasized to the lungs. Surprisingly, FGFBP1 expression level was 32 folds lower in SMC2 KD E0771 cells yet FGF2 was 16 folds up in the tumors from SMC2 KD E0771 cells. Tumor histology shows impaired and leaky angiogenesis in FGFBP1-/- compared to FGFBP1+/+ mice. Furthermore, higher levels of GM-CSF and G-CSF were detected in the conditioned media (CM) of SMC2 KD E0771 cells compared to control cells suggesting differential immune cell recruitment. Also, an endothelial monolayer exposed to CM from SMC2 KD E0771 cells showed delayed wound closure and loose junctions suggesting a tendency for endothelium disintegration and initiation of blood vessel formation. Interestingly, the CM effect was blocked using PD173074, a pan FGFR inhibitor. In conclusion, SMC2 demonstrate a role in regulating FGF signaling which affects the course of angiogenesis during tumor development. Note: This abstract was not presented at the conference. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Weiner M. Louis, Anna T. Riegel, Anton Wellstein. SMC2 role in regulating tumor angiogenesis via FGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A15.

Abstract B44: Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to large family of cysteine-rich proteins that is implicated in crosslinking keratin intermediate filaments during hair formation. To date, no role of these proteins in cancer has been reported. We found that depletion of Krtap5-5 from cancer cells led to cell blebbing, a loss of keratins 14 and 18 in addition to the upregulation of vimentin intermediate filaments. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extravasation of cancer cells into tissues in a zebrafish model of extravasation. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates cell motility and vascular invasion. Citation Format: Eric B. Berens, Ghada M. Sharif, Marcel O. Schmidt, Casey W. Shuptrine, Louis M. Weiner, Eric Glasgow, Anna T. Riegel, Anton Wellstein. Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B44.

Abstract 2689: Drivers of cancer cell invasion and metastasis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, evasion at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive and metastatic phenotype. Different human and mouse cancer cell lines were transduced with lentiviral-based, genome-wide or kinome-targeted shRNA libraries. Cancer cell populations of different invasive and metastatic phenotype were then selected for their loss- or gain-of-function respectively in endothelial invasion in vitro or by tumor growth and metastasis selection in vivo. After several rounds of selection, the respective shRNA targeted genes were identified from the selected cancer cell populations or from clonal cell lines. Eighteen different genes targeted by two to four distinct shRNAs each were identified in the in vitro selection for endothelial attachment and invasion. The function of the genes as well as the clinical significance appear to explain their phenotypic selection. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Louis M. Weiner, Zhang-Zhi Hu, Anna T. Riegel, Anton Wellstein. Drivers of cancer cell invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2013-2689