3D Colony Research Articles

Abstract 3975: PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy

Abstract BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) is a gene discovered in 2006 in the context of obesity, which we recently showed inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival of patients with gliomas and medulloblastomas (Erdreich-Epstein et al, Clin Cancer Res, In Press). PID1 mRNA was lower in more aggressive medulloblastomas and gliomas, and patients whose tumor had higher PID1 mRNA had longer survival. Moreover, overexpression of PID1 markedly decreased 2D colony formation in cell lines of medulloblastomas, GBM and atypical teratoid rhabdoid tumors (ATRT). The growth-inhibitory effect was due to both decreased proliferation and increased cell death. METHODS/RESULTS: COSMIC showed no mutations of PID1 in brain tumors, suggesting that PID1 was not a formal tumor suppressor gene. Therefore, we hypothesized that PID1 may sensitize brain tumor cells to therapy, and thus account for the longer survival in patients with higher PID1 mRNA. Indeed, while both cisplatin (10µg/ml) or transient PID1 overexpression increased apoptosis of glioma and medulloblastoma cell lines (indicated by increased AnnexinV and mitochondrial depolarization), combining cisplatin with PID1 caused a markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis (AnnexinV), suggesting that PID1 may be required for cisplatin-induced apoptosis. Taken together, this suggests a sensitizing effect of PID1 to chemotherapy in both glioma and medulloblastoma cell lines. Intriguingly, PID1 mRNA and protein both increased in response to etoposide (5µg/ml), vincristine (50ng/ml) or cisplatin (5µg/ml) in a time- and dose-dependent manner in the brain tumor cell lines. This chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFKB, suggesting that regulation of PID1 may be an NFKB-dependent mechanism. Ongoing work seeks to decipher the mechanism of these effects and test them in vivo in mouse models. CONCLUSIONS: Our data suggest that PID1 acts to sensitize glioma and medulloblastoma cells to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and survival in patients. Citation Format: Jingying Xu, Xiuhai Ren, Anthony Tran, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2014-3975

Large-Scale Parallel Simulations of 3D Cell Colony Dynamics

Biological processes are inherently complex and involve many unknown relationships and mechanisms at different scales. Despite many efforts, researchers still can't explain all of the observed phenomena and, if necessary, make any desirable changes in the dynamics. Recently, it has become apparent that a research opportunity lies in complementing the traditional, heuristic experimental approach with mathematical modeling and computer simulations. Achieving a simulation scale that corresponds, for instance, to clinically detectable tumor sizes is still a huge challenge, however, this scale is necessary to understand and control complex biological processes. This article presents a novel, high-performance computational approach allowing simulations of 3D cell colony dynamics at a previously unavailable tissue scale. Due to its high parallel scalability, the method achieves simulation of cell colonies composed of 109 cells, which allows for describing tumor growth in its early clinical stage.

Influence of individual cell motility on the 2D front roughness dynamics of tumour cell colonies.

The dynamics of in situ 2D HeLa cell quasi-linear and quasi-radial colony fronts in a standard culture medium is investigated. For quasi-radial colonies, as the cell population increased, a kinetic transition from an exponential to a constant front average velocity regime was observed. Special attention was paid to individual cell motility evolution under constant average colony front velocity looking for its impact on the dynamics of the 2D colony front roughness. From the directionalities and velocity components of cell trajectories in colonies with different cell populations, the influence of both local cell density and cell crowding effects on individual cell motility was determined. The average dynamic behaviour of individual cells in the colony and its dependence on both local spatio-temporal heterogeneities and growth geometry suggested that cell motion undergoes under a concerted cell migration mechanism, in which both a limiting random walk-like and a limiting ballistic-like contribution were involved. These results were interesting to infer how biased cell trajectories influenced both the 2D colony spreading dynamics and the front roughness characteristics by local biased contributions to individual cell motion. These data are consistent with previous experimental and theoretical cell colony spreading data and provide additional evidence of the validity of the Kardar-Parisi-Zhang equation, within a certain range of time and colony front size, for describing the dynamics of 2D colony front roughness.

Generating size-controlled embryoid bodies using laser direct-write

Embryonic stem cells (ESCs) have the potential to self-renew and differentiate into any specialized cell type. One common method to differentiate ESCs in vitro is through embryoid bodies (EBs), three-dimensional cellular aggregates that spontaneously self-assemble and generally express markers for the three germ layers, endoderm, ectoderm, and mesoderm. It has been previously shown that both EB size and 2D colony size each influence differentiation. We hypothesized that we could control the size of the EB formed by mouse ESCs (mESCs) by using a cell printing method, laser direct-write (LDW), to control both the size of the initial printed colony and the local cell density in printed colonies. After printing mESCs at various printed colony sizes and printing densities, two-way ANOVAs indicated that the EB diameter was influenced by printing density after three days (p = 0.0002), while there was no effect of the printed colony diameter on the EB diameter at the same timepoint (p = 0.74). There was no significant interaction between these two factors. Tukey's honestly significant difference test showed that high-density colonies formed significantly larger EBs, suggesting that printed mESCs quickly aggregate with nearby cells. Thus, EBs can be engineered to a desired size by controlling printing density, which will influence the design of future differentiation studies. Herein, we highlight the capacity of LDW to control the local cell density and colony size independently, at prescribed spatial locations, potentially leading to better stem cell maintenance and directed differentiation.

Abstract C274: FGF receptor mutations and fusions are therapeutic targets for inhibition by AZD4547.

Abstract Aberrant fibroblast growth factor (FGF) signaling has been implicated in tumorgenesis and chemoresistance in a number of cancer types including lung, breast, gastric, endometrial as well as others. The small molecule FGF receptor (FGFR) kinase inhibitor, AZD4547, is currently in Phase I/II clinical development (gastric, squamous lung and breast cancer) to test the FGFR amplified tumor-driven hypothesis. Our aim was to identify and characterize additional genetic subtypes based on reported FGFR aberrations and determine the sensitivity to AZD4547. Eight mutations in FGFR2: G614V, W290C, H544Q, D238N, N211I, T786K, K659N, and N550K and 3 mutations in FGFR3: S249C, R248C, Y373C, plus the FGFR fusions FGFR3-TACC3 and FGFR1-TACC1 found in ∼3% of glioblastoma [Singh et al., Science 337, 1231 (2012)] were assessed for in vitro transformation of normal breast epithelial cells (MCF10A) in a 3D soft agar colony formation assay. Several FGFR mutations/fusions were able to drive in vitro transformation as well as increase downstream signaling (pFGFR, pFRS2) in MCF10A engineered lines. For most FGFR mutations AZD4547 inhibited colony formation and downstream signaling at concentrations comparable to the WT isoforms (10-<1nM). Although AZD4547 inhibited N550K, a shift in potency was observed (>100nM). Taken together, these findings characterized gain-of-function mutations in FGFR2, FGFR3, and FGFR-TACC fusions and demonstrated the antiproliferative activity of AZD4547 against key genetic sub-types of FGFR aberrant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C274. Citation Format: Christopher R. Denz, Nin Guan, Kelly Jacques, Elaine Kilgour, Emma Jenkins, Sylvie Guichard, Paul Smith. FGF receptor mutations and fusions are therapeutic targets for inhibition by AZD4547. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C274.

ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

Aberrant regulation of the erythroblastosis oncogene B (ErbB) family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers. ErbB3 is required in luminal mammary epithelial cells (MECs) for growth and survival. Since breast cancer phenotypes may reflect biological traits of the MECs from which they originate, we tested the hypothesis that ErbB3 drives luminal breast cancer growth. We found higher ERBB3 expression and more frequent ERBB3 gene copy gains in luminal A/B breast cancers compared with other breast cancer subtypes. In cell culture, ErbB3 increased growth of luminal breast cancer cells. Targeted depletion of ErbB3 with an anti-ErbB3 antibody decreased 3D colony growth, increased apoptosis, and decreased tumor growth in vivo. Treatment of clinical breast tumors with the antiendocrine drug fulvestrant resulted in increased ErbB3 expression and PI3K/mTOR signaling. Depletion of ErbB3 in fulvestrant-treated tumor cells reduced PI3K/mTOR signaling, thus decreasing tumor cell survival and tumor growth. Fulvestrant treatment increased phosphorylation of all ErbB family RTKs; however, phospho-RTK upregulation was not seen in tumors treated with both fulvestrant and anti-ErbB3. These data indicate that upregulation of ErbB3 in luminal breast cancer cells promotes growth, survival, and resistance to fulvestrant, thus suggesting ErbB3 as a target for breast cancer treatment.

Double targeting of Survivin and XIAP radiosensitizes 3D grown human colorectal tumor cells and decreases migration

Background and purposeIn the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways. Materials and methodsColorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65). ResultsWhile basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing. ConclusionsOur results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration.

Abstract 1104: Malignant stroma regulates luminal breast cancer-cell growth versus basal breast cancer cell migration through different growth factor pathways.

Abstract The stromal microenvironment plays an important role in growth and aggressiveness of breast cancers. We have generated a reactive mouse mammary stromal cell line called BJ3Z that is tumorigenic in nude mice. In mixed-cell xenografts, BJ3Z cells enhance growth and angiogenesis of solid tumors established from MCF-7 human breast cancer cells. To minimize extensive dependence on in vivo studies, we have developed Matrigel-based 3D colony co-culture assays to analyze the role of the stromal microenvironment on epithelial-cell derived breast cancers in vitro. First, to define the properties of reactive BJ3Z cells, they were contrasted to normal mouse mammary gland fibroblasts (NMF), both grown in 3D. Expression profiling showed that Transforming Growth Factor-β1 (TGF-β1) and Platelet-Derived Growth Factor ligands A and B (PDGF-A & PDGF-B) are upregulated in BJ3Z cells. Next, we contrasted effects of BJ3Z and NMF cells on the three major breast cancer subtypes: Luminal MCF-7 cells; HER2 amplified BT-474 cells; Basal-like MDA-MB-231 and BT-20 cells. We find that malignant stroma has differential effects depending on the breast cancer subtype. 1. With regard to PDGF: Reactive stroma generated by BJ3Z cells in 3D, increases the growth of Luminal and HER2-amplified breast cancer cells as measured by BrdU incorporation. In contrast, BJ3Z cells suppress growth of Basal-like breast cancer cells. Similar results were obtained using BJ3Z-conditioned media. This suggests that the effects of BJ3Z cells on Luminal tumor-cell growth involve soluble factors. In that regard, use of Imatinib Mesylate (IM) a tyrosine kinase inhibitor against PDGF receptors completely blocks the rise in MCF-7 cell proliferation caused by BJ3Z-cell co-culture, or by BJ3Z cell-conditioned medium. In order to demonstrate that this is not due to a mouse-specific factor we showed that recombinant human PDGF-BB enhances 3D growth of MCF-7 cells. Parenthetically, BJ3Z-conditioned medium also enhances an in vitro angiogenesis model by a PDGF-dependent pathway that can be inhibited by IM. 2. With regard to TGF-β this pathway is not involved in Luminal tumor-cell growth. Rather in Basal-like cancer cells, BJ3Z or their conditioned medium use TGF-β pathway-dependent Smad 2/3 phosphorylation to enhance cell migration. This effect can be blocked by a TGF-βR1 inhibitor. We conclude that the stromal microenvironment uses the PDGF pathway to regulate Luminal tumor-cell growth and angiogenesis; the TGF-β pathway to regulate Basal tumor-cell aggressiveness. Citation Format: Mauricio P. Pinto, Britta M. Jacobsen, Kathryn B. Horwitz. Malignant stroma regulates luminal breast cancer-cell growth versus basal breast cancer cell migration through different growth factor pathways. [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 1104. doi:10.1158/1538-7445.AM2013-1104

Abstract 2174: Small molecule iInhibitors of PIM kinases as potential treatments for urothelial carcinomas.

Abstract The proto-oncogene PIM kinases (PIM-1, PIM-2, PIM-3) are serine/threonine kinases that have been shown to be involved in a number of signaling pathways important to cancer cells. PIM kinases act as downstream effectors as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, gastric, and head & neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma, and to assess for expression that may contribute to disease progression and serve as a potential site for targeted therapy. Seventy-two cases of urothelial carcinoma were included in this retrospective study of surgical biopsy and resection specimens from the University of Utah Department of Pathology (retrieved from 2008-2011). Tissue was stained with commercially available antibodies against PIM-1, PIM-2, and PIM-3. Cases were divided into three groups (invasive high grade urothelial carcinoma (n=49), non-invasive urothelial carcinoma/carcinoma in situ (n=16), and non-invasive low grade urothelial carcinoma (n=7)). Individual cases were then given a score (0-4) based upon a percentage of cells staining positive for each antibody (<5%=0; 5-25%=1; 26-50%=2; 51-75%=3; >75%=4). A score of 2 or greater was considered expressed. PIM-1, PIM-2 and PIM-3 expression was noted in 29% (2/7), 43% (3/7) and 86% (6/7) cases of non-invasive low-grade urothelial carcinoma; 44% (7/16), 50% (8/16), 44% (7/16) cases of non-invasive high-grade urothelial carcinoma; 10% (5/49), 27% (13/49), and 18% (9/49) cases of invasive high-grade urothelial carcinoma, respectively. These results suggest that expression of PIM-1, PIM-2 and PIM-3 is present in a significant percentage of urothelial carcinomas and may serve as a source for targeted PIM-kinase inhibition. We have developed PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family compared to our original PIM inhibitor SGI-1776. Our PIM inhibitors display sub-μM activity in pharmacodynamic marker modulation, proliferation and 2D colony formation assays using the UM-UC-3 bladder cancer cell line. These PIM kinase inhibitors also are potent inducers of apoptosis in T24, RT4, and UM-UC-3 bladder cancer cell lines. These compounds have favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using bladder cancer cell line models show that PIM kinase inhibition can reduce the tumor growth of these tumor models suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas. Citation Format: Kent J. Carpenter, Rachel Brog, Christopher Moreno, Daniel J. Albertson, Jared J. Bearss, Ting Liu, Steven Warner, David J. Bearss. Small molecule iInhibitors of PIM kinases as potential treatments for urothelial carcinomas. [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 2174. doi:10.1158/1538-7445.AM2013-2174

Surface Bound Amine Functional Group Density Influences Embryonic Stem Cell Maintenance

Gradient surfaces are highly effective tools to screen and optimize cell- surface interactions. Here, the response of embryonic stem (ES) cell colonies to plasma polymer gradient surfaces is investigated. Surface chemistry ranged from pure allylamine (AA) plasma polymer on one end of the gradient to pure octadiene (OD) plasma polymer on the other end. Optimal surface chemistry conditions for retention of pluripotency were identified. Expression of the stem cell markers alkaline phosphatase (AP) and Oct4 varied with the position of the ES cell colonies across the OD-AA plasma polymer gradient. Both markers were more strongly retained on the OD plasma polymer rich regions of the gradients. The observed variation of expression across the plasma polymer gradient increased with duration of stem cell culture. While maximum cell adhesion to the gradient substrate occurred at a nitrogen- to-carbon (N/C ratio) of approximately 0.1, Oct4 and AP expression was best retained at an N/C ratio < 0.04. Stem cell marker expression correlated with colony size and morphology: more compact, multilayered colonies with prominent F-actin staining arose as the N/C ratio decreased. Disruption of actin polymerization using Y-27632 ROCK inhibitor resulted in a collapse of the multilayer colony structure into monolayers with limited cell-cell contact. A corresponding decrease in expression of AP and Oct4 was observed. Oct4 expression along with 3D colony morphology was partially rescued on the OD plasma polymer rich regions of the gradient.

Homeodomain‐containing protein HOXB9 regulates expression of growth and angiogenic factors, facilitates tumor growth in vitro and is overexpressed in breast cancer tissue

HOXB9 is a homeobox-containing gene and is critical for the development of mammary gland and sternum. HOXB9 is also regulated by estrogen and is critical for angiogenesis. We investigated the biochemical roles of HOXB9 and its homeodomain in cell-cycle progression and tumorigenesis. Our studies demonstrated that HOXB9 is overexpressed in breast cancer tissue. HOXB9 overexpression stimulated 3D formation in soft agar assay. HOXB9 binds to the promoters of various tumor growth and angiogenic factors and regulates their expression. The homeodomain of HOXB9 plays crucial roles in transcriptional regulation of tumor growth factors and also in 3D colony formation, indicating crucial roles of the HOXB9 homeodomain in tumorigenesis. Overall, we demonstrated that HOXB9 is a critical regulator of tumor growth factors and is associated with tumorigenesis.

Abstract LB-257: Ganitumab (AMG 479), an anti IGF-1R antibody, inhibits the proliferation of melanoma cells

Abstract It has been shown that the Insulin-like growth factor receptor type I (IGF-1R) signaling pathway is highly active in metastatic melanoma. IGF-1 binding induces autophosphorylation of IGF-1R and initiation of MAPK and Akt pathways signaling which control cellular proliferation and survival. IGF-1 is a critical factor for growth of many types of cancer, including melanomas, and inhibition of its activity can trigger apoptosis by suppressing the PI3K-AKT and MAPK pathways. Our main objective in this study was to evaluate the therapeutic potential of a novel antibody to the IGF-1R (ganitumab) in a large panel of melanoma cancer cells. We have evaluated 41 human melanoma lines with well established genotypes. Among the 41 cell lines, 22 harbored the BRAF mutation, 10 were NRAS mutant and 9 were wild type for BRAF and NRAS mutations. Treatment with ganitumab (100µg/ml) resulted in inhibition of cell proliferation at 120 hrs in 20 out of 41 cell lines. We found that the response to ganitumab was independent of genotype. We chose 6 cell lines, 3 from the responder population and 3 non-responders (2 BRAF, 2 NRAS and 2 wild types) to further investigate the mechanism of action of ganitumab. Results from flow cytometry suggest that treatment of melanoma cells with 100 µg/ml ganitumab caused a 10% increase in apoptosis 72 hrs after treatment in sensitive cell lines compared with the resistant cell lines where we could not detect an increase in apoptotic cells. The effect of ganitumab was also investigated in a 3D colony soft agar formation assay. Melanoma cell lines sensitive to ganitumab showed a significant reduction in the ability to form colonies in soft agar compared with the cells that did not respond to the antibody. Ganitumab treatment led to a decrease in pAKT, pERK and pS6 in sensitive cell lines regardless of their driver oncogenic mutations when compared with non responder cell lines that showed no inhibition of these markers. In conclusion, inhibition of oncogenic MAPK and PI3K signaling by ganitumab resulted in activity in vitro against a large subset of melanoma cell lines. Our findings provide the rationale for further preclinical evaluation of ganitumab as a melanoma therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-257. doi:1538-7445.AM2012-LB-257

Abstract 3762: Second generation PIM inhibitors exhibit improved activity in solid tumor models

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) includes constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphomas, leukemias, multiple myeloma, prostate and bladder cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, and promoting tumor cell survival and proliferation. In the HEK-293T cell line, enhanced PIM kinase substrate BAD phosphorylation (pBAD) was observed following PIM and BAD overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, and more effectively by second generation PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-µM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the UM-UC-3 human bladder cancer cell line. PIM1 and PIM2 overexpression models were established in the human prostate cancer cell line 22RV-1 and the non-tumorigenic mouse NIH-3T3 cell background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In vivo xenograft studies using both PIM overexpression models and a clinically relevant solid tumor model facilitated identification of a lead candidate with demonstrated efficacy and favorable toxicity. IND-enabling studies with a lead candidate are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3762. doi:1538-7445.AM2012-3762

P2-02-06: Inhibition of CDK4 Phosphorylation of Smad3 Decreases Cyclin D Overexpressing Breast Cancer Cell Proliferation and Migration.

Abstract Introduction: Several aspects of breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their associated downstream signaling components, the Smads. Alterations in Smad signaling have been directly implicated in the dichotomous role of TGFβ in malignancy, enacting both tumor suppressant and tumor promoting behaviors in breast carcinogenesis. Our previous work characterized Smad3 as a tumor suppressor and found that Smad3 action is inhibited upon phosphorylation by cyclin D/CDK4 in breast cancer cells. Others have shown that CDK phosphorylation of Smad3 can affect cell motility. Based on these findings, we hypothesized that activation of CDK4 leads to phosphorylation and inhibition of Smad3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Methods: MCF7 parental and cyclin D overexpressing cells (MCF7 CD1) were treated with a CDK4 inhibitor alone or in combination with doxorubicin or paclitaxel and cell proliferation was determined using the MTS assay. MCF7 study cells were also transduced with lentiviral empty vector, wild-type (WT) Smad3, or Smad3 containing mutant CDK sites (T179V and 5M) resistant to inhibitory cyclin D/CDK phosphorylation. Stably transduced cells were plated in Matrigel 3-dimensional (3D) culture and treated with CDK4 inhibitor alone or with doxorubicin over a 12 day time course. Colony area was measured using MetaMorph software. Transwell migration and Matrigel invasion assays were used to determine the effect that transfection with Smad3 CDK phosphorylation site mutants or treatment with a CDK4 inhibitor had on highly metastatic cyclin D expressing MDA-MB-231 cells. Study wells were fixed/stained and the number of migrated or invaded cells was counted. Results: Treatment with the CDK4 inhibitor alone resulted in a growth inhibitory effect on proliferation of MCF7 parental and CD1 cells. Treatment with the CDK4 inhibitor in combination with chemotherapy resulted in the greatest inhibition of cell proliferation. In 3D culture, colonies formed by MCF7 study cells transduced with T179V or 5M Smad3 mutant constructs had smaller acinar size when compared with vector-transduced control cells. When compared to untreated cells, the MCF7 CD1 cells treated with CDK4 inhibitor or chemotherapy alone had smaller acinar size, with greatest decrease in size observed when the treatments were combined. Lastly, MDA-MB-231 cells transfected with the 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration and invasion, when compared with cells transfected with both vector control or WT Smad3. Treatment of the MDA-MB-231 cells with the CDK4 inhibitor also resulted in a significant decrease in cell migration and invasion. Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 resulted in a decrease in breast cancer cell proliferation, 3D colony formation and cell migration and invasion. Treatment with the CDK4 inhibitor in combination with chemotherapy further augmented these results. Inhibition of CDK4 and restoration of Smad3 activity may have a role in the treatment of breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-06.

Abstract C200: Second-generation PIM inhibitors exhibit improved activity in solid tumor models in vitro.

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) comprises constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, bladder, gastric and head &amp; neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, promoting tumor cell survival and proliferation. PIM-1 and PIM-2 overexpression models were obtained in the human prostate cancer cell lines PC-3M and 22RV-1 and the non-tumorigenic mouse NIH-3T3 background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In the PC-3M cell line, enhanced phosphorylation of the PIM kinase substrate BAD (pBAD) was observed following PIM overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, as well as next generation PIM inhibitors exhibiting 4–10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-μM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the PC-3M prostate cancer cell line, the UM-UC-3 bladder cancer cell line, and the HSC3 head &amp; neck cancer cell line. The second generation PIM inhibitors possess favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using both PIM overexpression models and clinically relevant solid tumor models will facilitate identification of a clinical candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C200.

Abstract 5370: PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression

Abstract The current study has addressed the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n=147) revealed PTEN expression to be lost in &amp;gt;10% of all melanoma cases. Although PTEN status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with little cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (&amp;gt;14-fold) compared to the PTEN- cell lines (4-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through re-introduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced both BIM expression and the level of apoptosis. The importance of rebound PI3K/AKT signaling in BRAF inhibitor resistance was confirmed in 3D organotypic cell culture and long-term colony formation assays. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2011-5370

Abstract 2431: Inhibition of metastasis through therapeutic targeting of focal adhesion kinase (FAK) in pancreatic cancer

Abstract BACKGROUND: Cancer mortality is primarily caused by metastatic disease; it has therefore been suggested that the preclinical evaluation of novel therapies should include testing in metastatic models of disease. This is especially relevant in light of recent reports of both conventional and targeted therapies being associated with accelerated metastases despite maintenance of primary tumor control. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which also acts as molecular scaffold, and localizes to focal adhesions, contact points between the cell and the extra-cellular matrix. Downstream of integrins and transmembrane receptors, FAK acts as a signaling hub, participating in processes integral to tumor progression including cell survival, proliferation, angiogenesis, migration and invasion. In this ongoing study, we explore the efficacy of selective genetic and pharmacologic FAK inhibition in preclinical models of human pancreatic cancer, including spontaneously metastasizing, cell-line derived and primary xenografts. METHODS &amp; RESULTS: We constructed modified pancreatic cancer cell lines using retroviral over-expression of the dominant negative FRNK (FAK related non-kinase). Pharmacologic inhibition of FAK was performed with PF-562271 (Pfizer), a selective FAK/Pyk2 inhibitor which recently completed Phase I testing. Adhesion, invasion and migration in vitro assays were conducted using the xCELLigence RTCA system. In vivo efficacy of FAK inhibition was evaluated in orthotopic xenograft models. In vitro studies demonstrated that both modalities of FAK inhibition resulted in impaired adhesion, invasion and migration and 3D colony formation, without comparable effects on viability, proliferation or colony formation in 2D. Expression of dominant-negative FRNK resulted in 49% smaller xenograft tumors (p=0.003), a 78% reduction in metastatic disease (p&amp;lt; 0.001) and longer overall survival (p &amp;lt; 0.001). There was a similar, 46% trend to reduction of metastatic burden (p=0.089) in orthotopic cell line xenograft models treated with PF-562271, despite a lack of significant primary tumor growth delay. CONCLUSIONS: Our data suggest a potential utility of selective FAK inhibition in delaying metastatic progression of pancreatic cancer. This is particularly relevant clinically since patients frequently present with locally advanced, unresectable disease and subsequently develop metastases. Pharmacologic FAK inhibition (in combination with conventional therapies) is currently being evaluated in neo/adjuvant models of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2011-2431

Abstract 3428: Coordinated regulation of mesenchymal epithelial transition in the PMC42-LA breast cancer cell line variant

Abstract PMC42 cells were derived from a breast cancer pleural effusion and exhibit stem cell-like features when first characterized (Whitehead, Bertoncello et al. 1983; Whitehead, Monaghan et al. 1983). The PMC42-LA subline (Ackland, Michalczyk et al. 2001; Ackland, Newgreen et al. 2003) is distinctly less mesenchymal than parental PMC42-ET cells, but both exhibit EGF- and TGFβ-inducible mesenchymal-like change. Together they provide an ideal model in which to study the regulation of epithelial-mesenchymal plasticity in a breast cancer context. We have assessed in vitro parameters including migration, 3D colony formation and EMT marker expression, and undertook both miRNA profiling (mirVana probe set V1, Ambion) and alternative splice usage (Affymetrix human 1.0 ST whole genome exon arrays) analysis. Several miRNAs were expressed differently in the two sublines and/or up- or down-regulated in response to EMT-inducing treatments. Relative to the mesenchymal ET subline, the, miR-200 family members were elevated in the epithelial LA subline, where a corresponding loss of Zeb1 expression was seen. The LA subline also showed reduced E-Cadherin promoter methylation, and increased methylation of the Zeb1 promoter. Manipulation of the miR 200 family affected the EMT marker expression in these cells, as did Zeb1 suppression with shRNA. Matrigel invasion was also inhibited with Zeb1 knockdown. Additional gene expression changes and alternative splice usage, promoter methylation changes, and miR expression changes are under study. These studies may identify molecules and pathways that are important in cell specification across the epithelial mesenchymal axis, and represent diagnostic and therapeutic targets in breast cancer. Ackland, M. L., A. Michalczyk, et al. (2001). “PMC42, A novel model for the differentiated human breast.” Exp Cell Res 263(1): 14-22. Ackland, M. L., D. F. Newgreen, et al. (2003). “Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells.” Lab Invest 83(3): 435-48. Whitehead, R. H., I. Bertoncello, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.” J Natl Cancer Inst 70(4): 649-61. Whitehead, R. H., P. Monaghan, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.” J Natl Cancer Inst 71(6): 1193-203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2011-3428

Investigations into the Metabolism of Two-Dimensional Colony and Suspended Microcarrier Cultures of Human Embryonic Stem Cells in Serum-Free Media

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10⁶ cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10⁶ cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.

Abstract 3372: Analysis of putative tumor stem cell markers in the NCI-60 tumor cell line panel

Abstract Tumor stem cells or cancer initiating cells are tumor cells that can regenerate a recurring tumor or a metastasis. A variety of cell surface and other markers have been associated with identification and characterization of tumor stem cells. We investigated the expression of a number of these putative stem cell markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP, and aldehyde dehydrogenase) in the NCI60 tumor panel. A spectrally compatible set of labeled antibodies was designed to enable simultaneous evaluation of six markers (CD44, CD24, CD133, CD166, CD326 (EpCAM), and PgP, and forward (FSC) and side scatter (SSC). Marker expression patterns were analyzed by multiple component analysis and correlated with clonogeneic potential of cell lines (2D colony formation assay, anchorage independent colony formation in soft agar assay). Additionally sphere formation in suspension culture and the capacity of cell lines to form tumors in vivo upon injection with matrigel was assessed for colon cancer cell lines. Analysis of expression of single markers and co-expression of up to 6 markers demonstrated that all stem cell markers investigated are expressed in cell lines of the NCI-60 panel. Patterns of co-expression of these markers as defined by multiple component analysis tracked primarily with tumor type, but individual patterns were also observed to occur in multiple tumor types. In the breast cancer panel the patterns of co-expression segregated with the known molecular sub-types (basal, luminal, normal-like) of the tumor cell lines. Cells from colonosphere cultures generated from the colon tumor panel using serum-free, growth factor supplemented medium showed subtly different co-expression patterns. Both sphere-derived and bulk cultured colon tumor cells were highly tumorigenic in NOD/SCID mice and tumor formation was observed following inoculation of 10-100 cells. Only two out of seven of the colonosphere derived cell populations had enhanced tumor initiating ability, but did not share differences in stem cell marker display with respect to the unselected bulk cultures. Furthermore, marker expression patterns did not significantly correlate with in vitro colony formation (2D culture, soft agar). Our results demonstrate a high degree of complexity and heterogeneity in tumor stem cell marker display that can be influenced by culture conditions and relates to the histological classification of tumors rather than to the clonogenic potential of cells in cell lines of the NCI60 panel. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3372.