Yoonseok Kam

Cadherin-Bound β-Catenin Feeds into the Wnt Pathway upon Adherens Junctions Dissociation: Evidence for an Intersection between β-Catenin Pools

β-catenin is an essential component of two cellular systems: cadherin-based adherens junctions (AJ) and the Wnt signaling pathway. A functional or physical connection between these β-catenin pools has been suggested in previous studies, but not conclusively demonstrated to date. To further examine this intersection, we treated A431 cell colonies with lysophosphatidic acid (LPA), which forces rapid and synchronized dissociation of AJ. A combination of immunostaining, time-lapse microscopy using photoactivatable-GFP-tagged β-catenin, and image analyses indicate that the cadherin-bound pool of β-catenin, internalized together with E-cadherin, accumulates at the perinuclear endocytic recycling compartment (ERC) upon AJ dissociation, and can be translocated into the cell nucleus upon Wnt pathway activation. These results suggest that the ERC may be a site of residence for β-catenin destined to enter the nucleus, and that dissociation of AJ may influence β-catenin levels in the ERC, effectively affecting β-catenin substrate levels available downstream for the Wnt pathway. This intersection provides a mechanism for integrating cell-cell adhesion with Wnt signaling and could be critical in developmental and cancer processes that rely on β-catenin-dependent gene expression.

Phospholipase D Activity Is Required for Actin Stress Fiber Formation in Fibroblasts

ABSTRACT Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ∼50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of α-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by α-actinin.

Exploiting evolutionary principles to prolong tumor control in preclinical models of breast cancer

Using evolutionary principles to guide drug administration, monotherapy with paclitaxel can maintain prolonged stability of breast cancer in preclinical models. Evolution of cancer therapy The standard approach to treating cancer is giving patients the maximum tolerated amount of chemotherapy with the goal of doing the maximum possible damage to the tumor without killing the patient. This method is relatively effective, but it also causes major toxicities. Now, Enriquez-Navas et al. have demonstrated a different approach for ensuring efficacy of chemotherapy and minimizing toxicity. The authors used an evolutionary approach, where the dose of chemotherapy is guided by the tumor’s response to the previous dose, allowing a gradual withdrawal of the drug if the tumor continues to respond. This method proved quite effective for paclitaxel treatment in two different mouse models and warrants further evaluation in additional models as well as human trials. Conventional cancer treatment strategies assume that maximum patient benefit is achieved through maximum killing of tumor cells. However, by eliminating the therapy-sensitive population, this strategy accelerates emergence of resistant clones that proliferate unopposed by competitors—an evolutionary phenomenon termed “competitive release.” We present an evolution-guided treatment strategy designed to maintain a stable population of chemosensitive cells that limit proliferation of resistant clones by exploiting the fitness cost of the resistant phenotype. We treated MDA-MB-231/luc triple-negative and MCF7 estrogen receptor–positive (ER+) breast cancers growing orthotopically in a mouse mammary fat pad with paclitaxel, using algorithms linked to tumor response monitored by magnetic resonance imaging. We found that initial control required more intensive therapy with regular application of drug to deflect the exponential tumor growth curve onto a plateau. Dose-skipping algorithms during this phase were less successful than variable dosing algorithms. However, once initial tumor control was achieved, it was maintained with progressively smaller drug doses. In 60 to 80% of animals, continued decline in tumor size permitted intervals as long as several weeks in which no treatment was necessary. Magnetic resonance images and histological analysis of tumors controlled by adaptive therapy demonstrated increased vascular density and less necrosis, suggesting that vascular normalization resulting from enforced stabilization of tumor volume may contribute to ongoing tumor control with lower drug doses. Our study demonstrates that an evolution-based therapeutic strategy using an available chemotherapeutic drug and conventional clinical imaging can prolong the progression-free survival in different preclinical models of breast cancer.

A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro

BackgroundClassical in vitro wound-healing assays and other techniques designed to study cell migration and invasion have been used for many years to elucidate the various mechanisms associated with metastasis. However, many of these methods are limited in their ability to achieve reproducible, quantitative results that translate well in vivo. Such techniques are also commonly unable to elucidate single-cell motility mechanisms, an important factor to be considered when studying dissemination. Therefore, we developed and applied a novel in vitro circular invasion assay (CIA) in order to bridge the translational gap between in vitro and in vivo findings, and to distinguish between different modes of invasion.MethodOur method is a modified version of a standard circular wound-healing assay with an added matrix barrier component (Matrigel™), which better mimics those physiological conditions present in vivo. We examined 3 cancer cell lines (MCF-7, SCOV-3, and MDA-MB-231), each with a different established degree of aggressiveness, to test our assays ability to detect diverse levels of invasiveness. Percent wound closure (or invasion) was measured using time-lapse microscopy and advanced image analysis techniques. We also applied the CIA technique to DLD-1 cells in the presence of lysophosphatidic acid (LPA), a bioactive lipid that was recently shown to stimulate cancer cell colony dispersal into single migratory cells, in order to validate our methods ability to detect collective and individual motility.ResultsCIA method was found to be highly reproducible, with negligible levels of variance measured. It successfully detected the anticipated low, moderate, and high levels of invasion that correspond to in vivo findings for cell lines tested. It also captured that DLD-1 cells exhibit individual migration upon LPA stimulation, and collective behavior in its absence.ConclusionGiven its ability to both determine pseudo-realistic invasive cell behavior in vitro and capture subtle differences in cell motility, we propose that our CIA method may shed some light on the cellular mechanisms underlying cancer invasion and deserves inclusion in further studies. The broad implication of this work is the development of a reproducible, quantifiable, high-resolution method that can be applied to various models, to include an unlimited number of parameters and/or agents that may influence invasion.

Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid

Mitogens activate protein translation through phosphorylation of p7S6 kinase (p70S6K) and eIF4E binding protein 1 (4E‐BP1) mediated by the mammalian target of rapamycin (mTOR) or phosphoinositide 3‐kinase (PI3K). A recent report (Science 294, 1942, 2001) has implicated phospholipase D (PLD) in mTOR signaling. We studied the role of PLD in the phosphorylation of p70S6K and 4E‐BP1 induced by lysophosphatidic acid (LPA) and platelet‐derived growth factor (PDGF) using fibroblasts deficient in PLD activity and also 1‐butanol, which inhibits phosphatidic acid production by PLD. The reduction in PLD activity in both situations impaired the effect of LPA on mTOR signaling but did not inhibit the effect of PDGF. PDGF induced marked phosphorylation of Akt (a PI3K target) but this was not affected by PLD deficiency. LPA caused much less phosphorylation of Akt and this was dependent on PLD activity. Toxin B, which inacti¬vates Rho GTPases, markedly impaired PLD1 activa¬tion and phosphorylation of Akt, p70S6K, and 4E‐BP1 induced by LPA but had a minimal or no effect on the actions of PDGF. These results support the hypothesis that LPA activates protein translation through the ac¬tion of PLD1‐generated PA on mTOR and the PI3K/ Akt pathway whereas PDGF acts through P13K/Akt independent of PLD1.—Kam, Y., Exton, J. H. Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid. FASEB J. 18, 311–319 (2004)

Evolutionary approaches to prolong progression-free survival in breast cancer

Many cancers adapt to chemotherapeutic agents by upregulating membrane efflux pumps that export drugs from the cytoplasm, but this response comes at an energetic cost. In breast cancer patients, expression of these pumps is low in tumors before therapy but increases after treatment. While the evolution of therapeutic resistance is virtually inevitable, proliferation of resistant clones is not, suggesting strategies of adaptive therapy. Chemoresistant cells must consume excess resources to maintain resistance mechanisms, so adaptive therapy strategies explicitly aim to maintain a stable population of therapy-sensitive cells to suppress growth of resistant phenotypes through intratumoral competition. We used computational models parameterized by in vitro experiments to illustrate the efficacy of such approaches. Here, we show that low doses of verapamil and 2-deoxyglucose, to accentuate the cost of resistance and to decrease energy production, respectively, could suppress the proliferation of drug-resistant clones in vivo. Compared with standard high-dose-density treatment, the novel treatment we developed achieved a 2-fold to 10-fold increase in time to progression in tumor models. Our findings challenge the existing flawed paradigm of maximum dose treatment, a strategy that inevitably produces drug resistance that can be avoided by the adaptive therapy strategies we describe.

Cellular modeling of cancer invasion: Integration of in silico and in vitro approaches

Cancer invasion is one of the hallmarks of cancer and a prerequisite for cancer metastasis. However, the invasive process is very complex, depending on multiple correlated intrinsic and environmental factors, and thus is difficult to study experimentally in a fully controlled way. Therefore, there is an increased demand for interdisciplinary integrated approaches combining laboratory experiments with multiscale in silico modeling. In this review, we will summarize current computational techniques applicable to model cancer invasion in silico, with a special focus on a class of individual‐cell‐based models developed in our laboratories. We also discuss their integration with traditional and novel in vitro experimentation, including new invasion assays whose design was inspired by computational modeling. J. Cell. Physiol. 227: 431–438, 2012.

Minimization of thermodynamic costs in cancer cell invasion

Metastasis, the truly lethal aspect of cancer, occurs when metastatic cancer cells in a tumor break through the basement membrane and penetrate the extracellular matrix. We show that MDA-MB-231 metastatic breast cancer cells cooperatively invade a 3D collagen matrix while following a glucose gradient. The invasion front of the cells is a dynamic one, with different cells assuming the lead on a time scale of 70 h. The front cell leadership is dynamic presumably because of metabolic costs associated with a long-range strain field that precedes the invading cell front, which we have imaged using confocal imaging and marker beads imbedded in the collagen matrix. We suggest this could be a quantitative assay for an invasive phenotype tracking a glucose gradient and show that the invading cells act in a cooperative manner by exchanging leaders in the invading front.

Dispersal of epithelial cancer cell colonies by lysophosphatidic acid (LPA)

We describe a model system in which cancer cell colonies disperse into single, highly migratory cells in response to lysophosphatidic acid (LPA). Though LPA is known to stimulate chemotaxis and chemokinesis, a colony dispersal effect has not been reported, to our knowledge. Cancer colony dispersal by LPA is comprised of an ordered sequence of events: (1) stimulation of membrane ruffling and formation of lamellipodia, (2) dissolution of adherens junctions, (3) single cell migration in a mesenchymal‐like morphology we term “ginkgo‐leaf.” The net result is dispersal of carcinoma cells from a compact colony. We analyzed these three steps using live‐cell imaging and computer‐assisted quantification and measured the following parameters: onset of lamellipodia formation, lamellipodia velocity, colony dispersal, trans‐epithelial resistance, migrating cell number and speed. Because hepatocyte growth factor (HGF) was described as an epithelial scatter factor, we compared it to LPA in our system and found that HGF has no epithelial colony dispersal properties and that this effect is strictly related to LPA. Given its striking similarity to tumor cell budding observed in patients, we propose that LPA‐colony dispersal may provide a cellular mechanism underlying cancer invasion and as such deserves further studies. J. Cell. Physiol. 206: 337–346, 2006.

Sweat but no gain: Inhibiting proliferation of multidrug resistant cancer cells with “ersatzdroges”

ATP‐binding cassette (ABC) drug transporters consuming ATPs for drug efflux is a common mechanism by which clinical cancers develop multidrug resistance (MDR). We hypothesized that MDR phenotypes could be suppressed by administration of “ersatzdroges,” nonchemotherapy drugs that are, nevertheless, ABC substrates. We reasoned that, through prolonged activation of the ABC pumps, ersatzdroges will force MDR cells to divert limited resources from proliferation and invasion thus delaying disease progression. We evaluated ABC substrates as ersatzdroge by comparing their effects on proliferation and survival of MDR cell lines (MCF‐7/Dox and 8226/Dox40) with the effects on the drug‐sensitive parental lines (MCF‐7 and 8226/s, respectively) in glucose‐limited condition. The changes in glucose and energy demands were also examined in vitro and in vivo. MCF‐7/Dox showed higher ATP demand and susceptibility to glucose resource limitation. Ersatzdroges significantly decreased proliferation of MCF‐7/Dox when the culture media contained physiological glucose concentrations (1.0 g/L) or less, but had no effect on MCF‐7. Similar evidence was obtained from 8226/Dox40 and 8226/s comparison. In vivo 18F‐FDG‐PET imaging demonstrated that glucose uptake was increased by systemic administration of an ersatzdroge in tumors composed of MDR. These results suggest that administration of ersatzdroges, by increasing the metabolic cost of resistance, can suppress proliferation of drug‐resistance phenotypes. This provides a novel and relatively simple application model of evolution‐based strategy, which can exploit the cost of resistance to delay proliferation of drug‐resistant cancer phenotypes. Furthermore, suggested is the potential of ersatzdroges to identify tumors or regions of tumors that express the MDR phenotype.