Theresa B. Phamduy

Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.

Laser direct-write of single microbeads into spatially-ordered patterns

Fabrication of heterogeneous microbead patterns on a bead-by-bead basis promotes new opportunities for sensors, lab-on-a-chip technology and cell-culturing systems within the context of customizable constructs. Laser direct-write (LDW) was utilized to target and deposit solid polystyrene and stem cell-laden alginate hydrogel beads into computer-programmed patterns. We successfully demonstrated single-bead printing resolution and fabricated spatially-ordered patterns of microbeads. The probability of successful microbead transfer from the ribbon surface increased from 0 to 80% with decreasing diameter of 600 to 45 µm, respectively. Direct-written microbeads retained spatial pattern registry, even after 10 min of ultrasonication treatment. SEM imaging confirmed immobilization of microbeads. Viability of cells encapsulated in transferred hydrogel microbeads achieved 37 ± 11% immediately after the transfer process, whereas randomly-patterned pipetted control beads achieved a viability of 51 ± 25%. Individual placement of >10 µm diameter microbeads onto planar surfaces has previously been unattainable. We have demonstrated LDW as a valuable tool for the patterning of single, micrometer-diameter beads into spatially-ordered patterns.

Laser Direct-Write Onto Live Tissues: A Novel Model for Studying Cancer Cell Migration

Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct‐write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333–2338, 2016.

The Power of CAD/CAM Laser Bioprinting at the Single-Cell Level: Evolution of Printing

The ability to deposit single cells in nonrestrictive two-dimensional and three-dimensional environments with high accuracy and reproducibility has proven invaluable for innovative cell and tissue research, and is now entering a new stage of maturity through systems engineering and next-generation designs. Coupling computer-aided design and computer-aided machining with laser direct write systems enables researchers to create reproducible biological models with micron-level precision. In particular, matrix-assisted pulsed-laser evaporation direct write system, and variations, has demonstrated this enhanced bottom-up approach to fabricating single-cell level constructs. This technology showcases the advantages of laser-assisted, nozzle-free, and contact-free printing that avoid probabilistic printing methods. In this chapter, we illustrate the importance of single-cell deposition and introduce laser-assisted bioprinting technology capable of micron-resolution and accuracy. This chapter will also review the post-deposition cell viability and mechanistics of laser-assisted transfer of biological materials.

Evaluation of electric cell-substrate impedance sensing for the detection of nanomaterial toxicity

Electric cell-substrate impedance sensing (ECIS) is an in situ and real-time monitoring system used to detect toxic agents by monitoring changes in impedance of a confluent cell monolayer. When toxic agents are introduced to cells, they can cause a change in the cell barrier function, a direct measure of the resistance to current flow caused by tight junction formation between cells. This exposure results in an immediate, quantitative change in the measured resistance between the electrodes, thus, continuously monitoring cell behaviour and by extension, toxic exposure. We have developed an ECIS-based protocol to functionally characterise epithelial cell response when challenged by different toxicants, particularly silver and copper nanoparticles. We verified our impedance changes with observed structural changes by fluorescent staining of zonula occludens-1 (ZO-1) protein in the tight junctions of a model epithelial cell line.

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.

Laser direct-write based fabrication of a spatially-defined, biomimetic construct as a potential model for breast cancer cell invasion into adipose tissue.

Epithelial-adipose interaction is an integral step in breast cancer cell invasion and progression towards lethal metastatic disease. Understanding the physiological contribution of obesity, a major contributor to breast cancer risk and negative prognosis in post-menopausal patients, on cancer cell invasion requires detailed co-culture constructs that reflect mammary microarchitecture. Using laser direct-write, a laser-based CAD/CAM bioprinting technique, we have demonstrated the ability to construct breast cancer cell-laden hydrogel microbeads into spatially defined patterns in hydrogel matrices containing differentiated adipocytes. Z-stack imaging confirmed the three-dimensional nature of the constructs, as well as incorporation of cancer cell-laden microbeads into the adipose matrix. Preliminary data was gathered to support the construct as a potential model for breast cancer cell invasion into adipose tissue. MCF-7 and MDA-MB-231 breast cancer cell invasion was tracked over 2 weeks in an optically transparent hydrogel scaffold in the presence of differentiated adipocytes obtained from normal weight or obese patient tissue. Our model successfully integrates adipocytes and gives us the potential to study cellular and tissue-level interactions towards the early detection of cancer cell invasion into adipose tissue.

Abstract 1596: Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Hope E. Burks, Margarite Matossian, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Lowry Curley, Muralidharan Anbalagan, Brian G. Rowan, Doug Chrisey, Jane E. Cavanaugh, Patrick T. Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1596.

Abstract 4410: ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway

Breast cancer is the most commonly diagnosed cancer in women, with the second highest mortality rate. The overwhelming majority of breast cancer deaths are a direct result of distant metastatic spread to vital organs such as lung, bone, and brain. The zinc finger transcription factor ZEB2 has been implicated as a driver of cancer cell motility, tissue invasion and metastasis in a number of diverse malignancies, but its role in breast cancer is not completely understood. We chose to examine the effects of direct overexpression of ZEB2 in the MCF-7 cell line, a luminal and estrogen receptor positive (ER+) derived breast cancer line. MCF-7-ZEB2 cells demonstrated significantly increased migration and invasion as well as an altered morphology in vitro compared to that of vector. Additionally, MCF-7-ZEB2 cells exhibited increased lung metastasis in vivo when implanted in an orthotopic xenograft mouse model. ZEB2 function in metastasis has canonically been attributed to transcriptional repression of the cell junction protein E-Cadherin. Here we establish that in ER+ breast cancer cells, ZEB2 fails to repress E-cadherin and promotes cell motility and metastasis through the induction of an E-cadherin independent signaling cascade. Next generation RNA sequencing analysis of the MCF-7-ZEB2 cells compared to vector revealed alteration of the MAPK signaling cascade, evidenced by enhanced expression of key motility and MAPK associated genes, including PLAU, EGF, ACTA2, and MMP9. Pharmacological inhibition of MAPK pathway completely abrogated ZEB2 induced migration, cell morphology changes and expression of target motility genes, confirming a necessary role for MAPK signaling in ZEB2-driven cell motility in ER+ breast cancer. Together these results indicate that the ZEB2 transcription factor drives motility in breast cancer cells through a novel MAPK dependent pathway, warranting further investigation into the mechanisms involved in ZEB2 action. Elucidating the pathways involved in ZEB2 function which are specific to ER+ breast cancer is an important step in understanding the processes underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay V. Rhodes, Elizabeth C. Martin, Van T. Hoang, Steven Elliott, Melody Badoo, Theresa Phamduy, Aaron Buechlein, Douglas Rusch, Douglas Chrisey, Erik Flemington, Kenneth Nephew, Bridgette Collins-Burow, Matthew E. Burow. ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4410.

Abstract 1052: Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition

The mitogen-activated protein kinase (MAPK) pathway has well-established roles in cellular processes including proliferation, differentiation, and regulation of cell fate, namely survival and apoptosis. In breast cancer, constitutive activation of the MAPK/extracellular signal-regulated kinases (ERK) pathways have been linked to chemoresistance and metastatic progression through distinct mechanisms including the activation of epithelial-to-mesenchymal transition (EMT). Our previous studies have shown that overexpression of MEK5 promotes EMT markers and induces the progression to a mesenchymal phenotype. Here, we tested the effects of a novel MEK1/2 and MEK5 inhibitor, SC-1-151, and other known MAPK signaling inhibitors (PD184,352 (MEK1/2), AZD6244 (MEK1/2), BIRB796 (p38)) on a panel of mesenchymal and highly metastatic breast cancer cell lines. While the MEK1/2 and p38 inhibitors decreased cell viability across cell lines, only the dual inhibition of MEK1/2 and MEK5 though the use of SC-1-151 demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET). Furthermore, the cells exhibited a significant decrease in migration potential following SC-1-151 treatment. Further analysis of the effects of SC-1-151 in the triple-negative breast cancer cell lines revealed an alteration of the genes associated with EMT, notably a decrease in expression of Fra-1, a transcription factor downstream of MAPK. Immuno-compromised mice inoculated with the MDA-MD-231 cell line and treated with SC-1-151 demonstrated decreased tumor volumes compared to vehicle-treated animals at day 30 post cell injection, implicating the role of MEK inhibition on tumorigenesis. These data demonstrate the need for a better understanding of the dual role of MEK1/2 and MEK5 signaling in breast cancer, and suggest that inhibition of the MEK1/2 and MEK5 signaling pathways leads to a decrease in EMT and cell migration. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Henry C. Segar, Hope Burks, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Doug Chrisey, Jane E. Cavanaugh, Patrick Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition. [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 1052. doi:10.1158/1538-7445.AM2014-1052