Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Targeting nuclear factor-kappa B to overcome resistance to chemotherapy

Intrinsic or acquired resistance to chemotherapeutic agents is a common phenomenon and a major challenge in the treatment of cancer patients. Chemoresistance is defined by a complex network of factors including multi-drug resistance proteins, reduced cellular uptake of the drug, enhanced DNA repair, intracellular drug inactivation, and evasion of apoptosis. Pre-clinical models have demonstrated that many chemotherapy drugs, such as platinum-based agents, antracyclines, and taxanes, promote the activation of the NF-κB pathway. NF-κB is a key transcription factor, playing a role in the development and progression of cancer and chemoresistance through the activation of a multitude of mediators including anti-apoptotic genes. Consequently, NF-κB has emerged as a promising anti-cancer target. Here, we describe the role of NF-κB in cancer and in the development of resistance, particularly cisplatin. Additionally, the potential benefits and disadvantages of targeting NF-κB signaling by pharmacological intervention will be addressed.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Distinguishing between mechanisms of cell aggregation using pair-correlation functions

Many cell types form clumps or aggregates when cultured in vitro through a variety of mechanisms including rapid cell proliferation, chemotaxis, or direct cell-to-cell contact. In this paper we develop an agent-based model to explore the formation of aggregates in cultures where cells are initially distributed uniformly, at random, on a two-dimensional substrate. Our model includes unbiased random cell motion, together with two mechanisms which can produce cell aggregates: (i) rapid cell proliferation, and (ii) a biased cell motility mechanism where cells can sense other cells within a finite range, and will tend to move towards areas with higher numbers of cells. We then introduce a pair-correlation function which allows us to quantify aspects of the spatial patterns produced by our agent-based model. In particular, these pair-correlation functions are able to detect differences between domains populated uniformly at random (i.e. at the exclusion complete spatial randomness (ECSR) state) and those where the proliferation and biased motion rules have been employed - even when such differences are not obvious to the naked eye. The pair-correlation function can also detect the emergence of a characteristic inter-aggregate distance which occurs when the biased motion mechanism is dominant, and is not observed when cell proliferation is the main mechanism of aggregate formation. This suggests that applying the pair-correlation function to experimental images of cell aggregates may provide information about the mechanism associated with observed aggregates. As a proof of concept, we perform such analysis for images of cancer cell aggregates, which are known to be associated with rapid proliferation. The results of our analysis are consistent with the predictions of the proliferation-based simulations, which supports the potential usefulness of pair correlation functions for providing insight into the mechanisms of aggregate formation.

In vitro assessment of migratory behavior of two cell populations in a simple multichannel microdevice

Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver “death-signals” to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards each other. Confluent monolayers of MSC and MCF-7 were established in adjacent chambers separated by an array of 14 microchannels. Initially, culture chambers were isolated by air bubbles (air-valves) contained within each microchannel, and then bubbles were displaced to initiate the assay. The MCF-7 cells migrated preferentially towards MSC, whilst the MSC did not migrate preferentially towards the MCF-7 cells. Our results corroborate previous literature that suggests MSC migration towards cancer cells in vivo is in response to the associated inflammation rather than directly to signals secreted by the cancer cells themselves.

methoxy-poly(ethylene glycol) modified poly(L-lactide) enhanced cell affinity of human bone marrow stromal cells by the upregulation of 1-cadherin and delta-2-catenin

Poly(l-lactide) (PLLA), a versatile biodegradable polymer, is one of the most commonly-used materials for tissue engineering applications. To improve cell affinity for PLLA, poly(ethylene glycol) (PEG) was used to develop diblock copolymers. Human bone marrow stromal cells (hBMSCs) were cultured on MPEG-b-PLLA copolymer films to determine the effects of modification on the attachment and proliferation of hBMSC. The mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analyzed using RT-qPCR to understand the underlying mechanisms. It was found that MPEG-b-PLLA copolymer films significantly improved cell adhesion, extension, and proliferation.This was found to be related to the significant upregulation of two adhesion genes, CDH1 and CTNND2, which encode 1-cadherin and delta-2-catenin, respectively, two key components for the cadherin-catenin complex. In summary, MPEG-b-PLLA copolymer surfaces improved initial cell adhesion by stimulation of adhesion molecule gene expression.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

The heat shock protein 90 inhibitor, 17-allylamino-17- demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor κB ligand (BANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence causedby 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

ICI 164,384, a pure antagonist of estrogen-stimulated MCF-7 cell proliferation and invasiveness

Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenylethylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, for their effects on the proliferation and invasiveness of the MCF-7 cell line and its antiestrogen-resistant variant LY-2. While all three antiestrogens blocked the proliferative effects of 17β-estradiol on MCF-7 cells, OHT and LY 117018, but not ICI 164,384 stimulated their proliferation in the absence of estrogen. The proliferative effects of OHT and LY 117018 were blocked by ICI 164,384. Basement membrane invasiveness of MCF-7 cells was stimulated by 17β-estradiol and OHT, but not LY 117018 or ICI 164,384. Both ICI 164,384 and Ly 117018 were able to block the invasiveness induced by either 17β-estradiol or OHT. The LY-2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proliferation, and responded only slightly to estrogen. ICI 164,384, but not OHT or LY 117018 antagonized the effects of 17β-estradiol, but did not reduce proliferation below control levels. The LY-2 line was not resistant to the antiestrogenic effects of LY 117018 or ICI 164,384 on invasiveness, and was stimulated by LY 117018 for this parameter. Thus, ICI 164,384 is a pure antiestrogen for MCF-7 cell proliferation and invasiveness, and may offer clinical advantage over nonsteroidal antiestrogens which can stimulate these activities in tumor models in vitro.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

Epithelial mesenchymal transition traits in human breast cancer cell lines

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.