Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells

Background:

Src family kinases control multiple cancer cell properties including cell cycle progression, survival, and metastasis. Recent studies suggest that the Src inhibitor dasatinib blocks these critical cancer cell functions.

Methods:

Because the molecular mechanism of action of dasatinib in breast cancers has not been investigated, we evaluated the effects of dasatinib as a single agent and in combination with the commonly used chemotherapeutic doxorubicin, on the proliferation, viability, and invasive capacity of breast cancer cells lines earlier categorised as dasatinib-sensitive (MDA-MB-231) and moderately resistant (MCF7 and T47D). We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.

Results:

The cell lines tested varied widely in sensitivity to growth inhibition (IC₅₀=0.16–12.3 μM), despite comparable Src kinase inhibition by dasatinib (IC₅₀=17–37 nM). In the most sensitive cell line, MDA-MB-231, dasatinib treatment induced significant G₁ accumulation with little apoptosis, disrupted cellular morphology, blocked migration, inhibited invasion through Matrigel (P<0.01), and blocked the formation of invadopodia (P<0.001). Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.

Conclusion:

The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.     

Hypoxia, Snail and incomplete epithelial�Cmesenchymal transition in breast cancer

Background:

Hypoxia is an element of the tumour microenvironment that impacts upon numerous cellular factors linked to clinical aggressiveness in cancer. One such factor, Snail, a master regulator of the epithelial–mesenchymal transition (EMT), has been implicated in key tumour biological processes such as invasion and metastasis. In this study we set out to investigate regulation of EMT in hypoxia, and the importance of Snail in cell migration and clinical outcome in breast cancer.

Methods:

Four breast cancer cell lines were exposed to 0.1% oxygen and expression of EMT markers was monitored. The migratory ability was analysed following Snail overexpression and silencing. Snail expression was assessed in 500 tumour samples from premenopausal breast cancer patients, randomised to either 2 years of tamoxifen or no adjuvant treatment.

Results:

Exposure to 0.1% oxygen resulted in elevated levels of Snail protein, along with changes in vimentin and E-cadherin expression, and in addition increased migration of MDA-MB-468 cells. Overexpression of Snail increased the motility of MCF-7, T-47D and MDA-MB-231 cells, whereas silencing of the protein resulted in decreased migratory propensity of MCF-7, MDA-MB-468 and MDA-MB-231 cells. Moreover, nuclear Snail expression was associated with tumours of higher grade and proliferation rate, but not with disease recurrence. Interestingly, Snail negativity was associated with impaired tamoxifen response (P=0.048).

Conclusions:

Our results demonstrate that hypoxia induces Snail expression but generally not a migratory phenotype, suggesting that hypoxic cells are only partially pushed towards EMT. Furthermore, our study supports the link between Snail and clinically relevant features and treatment response.     

A chemical biology screen reveals a role for Rab21-mediated control of actomyosin contractility in fibroblast-driven cancer invasion

Background:

Carcinoma-associated fibroblasts (CAFs) can promote the progression of tumours in many ways. They can remodel the extracellular matrix to generate an environment that enables the invasion of cancer cells. We hypothesised that compounds that prevent matrix remodelling by CAFs would block their ability to promote carcinoma cell invasion.

Methods:

We designed a screen for compounds that interfere with CAF-promoted matrix remodelling. Hits from this screen were investigated in organotypic invasion models of squamous cell carcinoma (SCC).

Results:

We find that lovastatin and simvastatin reduce matrix remodelling by fibroblasts and thereby reduce SCC invasion. This class of compounds exert their effects partly through disrupting the function of Rab proteins, and we show a new role for Rab21 in promoting cancer cell invasion promoted by CAFs.

Conclusions:

Rab21 is required for CAFs to promote the invasion of cancer cells. It enables the accumulation of integrin α5 at the plasma membrane and subsequent force-mediated matrix remodelling.     

ADAM10: a new player in breast cancer progression?

Background:

The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes.

Methods:

ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration.

Results:

Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer.

Conclusions:

Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.     

Cancer-associated fibroblasts induce epithelial–mesenchymal transition of breast cancer cells through paracrine TGF-β signalling

Background:

Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.

Methods:

Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.

Results:

The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell–extracellular matrix adhesion, migration and invasion, and promoted epithelial–mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.

Conclusion:

Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.     

Inhibitory effect of a TGF�� receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells

Background:

Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.

Methods:

Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.

Results:

TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.

Conclusion:

A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.     

CD44 targets Na+/H+ exchanger 1 to mediate MDA-MB-231 cells' metastasis via the regulation of ERK1/2

Background:

CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.

Methods:

The downregulation of CD44 was determined by immunofluorescence. Moreover, the motility of breast cancer cells was detected by wound-healing and transwell experiments. Then the spontaneous metastasis of CD44-silenced MDA-MB-231 cells was tested by histology with BALB/c nude mice.

Results:

A positive correlation between CD44 and Na⁺/H⁺ exchanger isoform 1 (NHE1) was found in two breast cancer cells. CD44 downregulation could inhibit the metastasis of MDA-MB-231 cells and the expressions of Na⁺/H⁺ exchanger 1. Moreover, CD44 overexpression upregulated the metastasis of MCF-7 cells, but the elevated metastatic ability was then inhibited by Cariporide. Interestingly, during these processes only the p-ERK1/2 was suppressed by CD44 downregulation and the expression of matrix metalloproteinases and metastatic capacity of MDA-MB-231 cells were greatly inhibited by the MEK1 inhibitor PD98059, which even had a synergistic effect with Cariporide. Furthermore, CD44 downregulation inhibits breast tumour outgrowth and spontaneous lung metastasis.

Conclusions:

Taken together, this work indicates that CD44 regulates the metastasis of breast cancer cells through regulating NHE1 expression, which could be used as a novel strategy for breast cancer therapy.     

Effects of ulinastatin and docetaxel on breast cancer invasion and expression of uPA,uPAR and ERK

Objective

To investigate the effects of ulinastatin and docetaxel on invasion of breast cancer cells and expression of uPA, uPAR and ERK, breast cancer MDA-MB-231 and MCF-7 cells.

Methods

The nude mice were treated with PBS, ulinastatin, docetaxel, and ulinastatin plus docetaxel, respectively. Their effects on 1) cell invasion ability was assayed using Transwell; 2) expression of uPA, uPAR and ERK was detected by real time PCR and Western blot; 3) uPA, uPAR and p-ERK protein level in nude mice was quantified by immunohistochemistry.

Results

1) Treatment with ulinastatin, docetaxel, and ulinastatin plus docetaxel, respectively, significantly inhibited MDA-MB-231 and MCF-7 cell invasion; 2) mRNA and protein levels of uPA, uPAR and ERK1/2 were inhibited by ulinastatin, but enhanced by docetaxel.

Conclusion

Ulinastatin can enhance the effects of docetaxel on invasion of breast cancer cells. And that uPA, uPAR and p-ERK expression is obviously inhibited by ulinastatin.     

A systematic review to establish the frequency of cyclooxygenase-2 expression in normal breast epithelium, ductal carcinoma in situ, microinvasive carcinoma of the breast and invasive breast cancer

Background:

Epidemiological studies have suggested a protective effect of cyclooxygenase (COX)-inhibiting non-steroidal anti-inflammatory drugs in breast cancer risk and disease progression. We performed a systematic review to evaluate the frequency of COX-2 expression in normal breast epithelium, ductal carcinoma in situ of breast (DCIS), DCIS-adjoining invasive breast cancer, microinvasive carcinoma of the breast (MICB) and invasive breast cancer.

Methods:

Literature searches were carried out on MEDLINE, EMBASE and Web of Science from their commencement until September 2010. Primary studies examining COX-2 expression by immunohistochemistry methodology were included. Meta-analyses were carried out using random effects models for individual study estimates of COX-2 expression and pooled to give an overall estimate.

Results:

The pooled prevalences (95% confidence intervals) of COX-2 expressions were 53% (44–61) in DCIS studies and 42% (36–49) in the invasive breast cancer studies. There were too few studies involving normal breast epithelium, DCIS-adjoining invasive breast cancer and MICB to conduct meta-analyses.

Conclusion:

The findings from our meta-analyses have shown similar COX-2 expression in DCIS and invasive breast cancer. This may suggest the involvement of COX-2 in early carcinogenesis. Further studies of COX-2 expression in DCIS are required to investigate the use of COX-2 as a potential drug target for prevention of disease progression in DCIS.     

Expression of COX-2, NF-��B-p65, NF-��B-p50 and IKK�� in malignant and adjacent normal human colorectal tissue

BACKGROUND:

Cyclooxygenase-2 (COX-2) is selectively over-expressed in colorectal tumours. The mechanism of COX-2 induction in these tumours is not fully understood, although evidence suggests a possible link between nuclear factor (NF)-κB and COX-2. We hypothesised an association between COX-2 expression and NF-κB-p65, NF-κB-p50 and IκB-kinase-α (IKKα) in both epithelial and stromal cells in human colorectal cancer.

Methods:

Using immunohistochemistry, we measured COX-2, NF-κB-p65, NF-κB-p65 nuclear localisation sequence (NLS), NF-κB-p50, NF-κB-p50 NLS and IKKα protein expression in matched colorectal biopsy samples comprising both non-tumour and adjacent tumour tissue from 32 patients with colorectal cancer.

Results:

We have shown that stromal cells of malignant and surrounding normal colorectal tissue express COX-2. In all cell types of malignant tissue, and in vascular endothelial cells (VECs) of neighbouring normal tissue, COX-2 expression was strongly associated with NF-κB-p65 expression (Pearson''s correlation, P=0.019 for macrophages, P=0.001 for VECs, P=0.002 for fibroblasts (malignant tissue), and P=0.011 for VECs (non-malignant tissue)) but not NF-κB-p50 or IKKα.

Conclusions:

These data suggest that in these cells COX-2 induction may be mediated through activation of the canonical NF-κB pathway. Finally, the lack of association between COX-2, NF-κB-p65 or IKKα in stromal cells with the clinical severity of colorectal cancer as determined by Duke''s stage, suggests that COX-2, NF-κB-p65 and IKKα expression are possibly early post-initiation events, which could be involved in tumour progression.     

Regulation of the apoptosis-inducing kinase DRAK2 by cyclooxygenase-2 in colorectal cancer

Background:

Cyclooxygenase-2 (COX-2) is over-expressed in colorectal cancer (CRC), rendering tumour cells resistant to apoptosis. Selective COX-2 inhibition is effective in CRC prevention, although having adverse cardiovascular effects, thus focus has shifted to downstream pathways.

Methods:

Microarray experiments identified genes regulated by COX-2 in HCA7 CRC cells. In vitro and in vivo regulation of DRAK2 (DAP kinase-related apoptosis-inducing kinase 2 or STK17β, an apoptosis-inducing kinase) by COX-2 was validated by qRT-PCR.

Results:

Inhibition of COX-2 induced apoptosis and enhanced DRAK2 expression in HCA7 cells (4.4-fold increase at 4 h by qRT-PCR, P=0.001), an effect prevented by co-administration of PGE₂. DRAK2 levels were suppressed in a panel of human colorectal tumours (n=10) compared to normal mucosa, and showed inverse correlation with COX-2 expression (R=−0.68, R²=0.46, P=0.03). Administration of the selective COX-2 inhibitor rofecoxib to patients with CRC (n=5) induced DRAK2 expression in tumours (2.5-fold increase, P=0.01). In vitro silencing of DRAK2 by RNAi enhanced CRC cell survival following COX-2 inhibitor treatment.

Conclusion:

DRAK2 is a serine–threonine kinase implicated in the regulation of apoptosis and is negatively regulated by COX-2 in vitro and in vivo, suggesting a novel mechanism for the effect of COX-2 on cancer cell survival.     

ING1b-inducible microRNA203 inhibits cell proliferation

Background:

The ING family of type II tumour suppressors serve as both epigenetic ‘readers'' and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) ‘writers'' of the epigenetic histone code. The ING1 protein has also been implicated in regulating microRNA (miRNA) levels. In this study, we identify a link between ING1b and the miRNA epigenetic network.

Methods:

Primary fibroblasts infected with adenoviruses expressing GFP control or GFP plus ING1b were examined for alterations in miRNA profiles using a miRNA PCR array. Additional experiments confirmed specificity and consequences of altered miRNA expression.

Results:

MicroRNAs miR-203, miR-375, miR-449b and miR-200c were increased by ING1b overexpression. Ectopic expression of miR-203 inhibited U2OS and MDA-MB-231 cancer cell growth, and induced G1 cell cycle arrest in U2OS cells as estimated by flow cytometry. Transfection with miR-203 inhibitor reversed the proliferation inhibition induced by ING1b in U2OS cells. CHIP assays showed that ING1b bound to the promoter of miR-203. Western blot analyses showed that CDK6, c-Abl and Src were downregulated by the transfection of miR-203.

Conclusion:

These results indicate that ING1b epigenetically regulates several miRNAs including miR-203. The several-fold increase in miR-203 by ING1b might inhibit cancer cell proliferation through coordinate downregulation of CDK6, c-Abl and Src.     

Enhancement of osteoclastogenic activity in osteolytic prostate cancer cells by physical contact with osteoblasts

Background:

The interaction between prostate cancer cells and osteoblasts is critical for the development of bone metastasis. Metastatic cancer cells may physically contact osteoblasts in the bone microenvironment; however, the biological significance of this interaction is not fully understood.

Methods:

Human prostate cancer cells (the osteolytic cell line PC-3 and the osteoblastic cell line MDA-PCa 2b) and human osteoblasts (hFOB1.19) were cocultured under two different conditions (bilayer and contact conditions). Differential gene expression profiles of prostate cancer cells were then investigated using microarray analysis. Differentially expressed genes were analysed using RT–PCR and western blotting, and the effect of anti-cadherin neutralising antibodies on their expression was assayed. The osteoclastogenic activity of cells grown under these different conditions was also investigated using an in vitro assay.

Results:

When PC-3 or MDA-PCa 2b cells were cocultured with hFOB1.19 cells under contact conditions, the expression of eight genes was upregulated and that of one gene was downregulated in PC-3 cells compared with gene expression in bilayer culture. No differentially expressed genes were detected in MDA-PCa 2b cells. Four of the eight upregulated genes (interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), IL-6 and the third component of complement (C3)) have already been reported to participate in osteoclastogenesis. Indeed, a cell lysate of PC-3 cells grown under contact coculture conditions significantly enhanced osteoclastogenesis in vitro (P<0.005). neutralisation of cadherin-11 with a specific antibody inhibited upregulation of COX-2 and C3 mRNA in PC-3 cells. In contrast, neutralisation of N-cadherin induced upregulation of COX-2 mRNA.

Conclusion:

Physical contact between osteolytic prostate cancer cells and osteoblasts may upregulate osteoclastogenesis-related gene expression in prostate cancer cells and enhance osteoclastogenesis. Additionally, cadherin-11 and N-cadherin are involved in this process. These data provide evidence supporting new therapies of prostate cancer bone metastasis that target direct cancer-cell-osteoblast cell–cell contact.     

Disulfiram targets cancer stem-like cells and reverses resistance and cross-resistance in acquired paclitaxel-resistant triple-negative breast cancer cells

Background:

Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic, the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients.

Methods:

In this study, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)–isobologram, western blot, ALDEFLUOR analysis, clonogenic assay and immunocytochemistry were used.

Results:

The chemoresistant MDA-MB-231_PAC10 cells are highly cross-resistant to paclitaxel (PAC), cisplatin (CDDP), docetaxel and doxorubicin. The MDA-MB-231_PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21^Waf1. The MDA-MB-231_PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins, for example, Oct4, Sox2, Nanog and nuclealisation of HIF2α and NF-κBp65. We have previously reported that disulfiram (DS), an antialcoholism drug, targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231_PAC10 cells.

Conclusion:

Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic, DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance.     

TNF-receptor-1 adaptor protein FAN mediates TNF-induced B16 melanoma motility and invasion

Background:

Locomotion of cancer cells can be induced by TNF and other motogenic factors secreted by cells of the tumour microenvironment such as macrophages. Based on our recent findings that the TNF receptor adaptor protein FAN mediates TNF-induced actin reorganisation and regulates the directed migration of immune cells responding to chemotactic cues, we addressed the role of FAN in cancer cell motility and the formation of invadopodia, a crucial feature in tumour invasion.

Methods:

In B16 mouse melanoma cells, FAN was downregulated and the impact on FAN on cell motility and invasion was determined using in vitro assays and in vivo animal models.

Results:

Like FAN^−/− murine embryonic fibroblasts, FAN-deficient B16 melanoma cells showed defective motility responses to TNF in vitro. In vivo FAN-deficient B16 melanoma cells produced significantly less disseminated tumours after i.v. injection into mice. Danio rerio used as a second in vivo model also revealed impaired spreading of FAN-deficient B16 melanoma cells. Furthermore, FAN mediated TNF-induced paxillin phosphorylation, metalloproteinase activation and increased extracellular matrix degradation, the hallmarks of functionally active invadopodia.

Conclusion:

The results of our study suggest that FAN through promoting melanoma cellular motility and tumour invasiveness is critical for the tumour-promoting action of TNF.