Two novel human anti-ErbB2 immunoagents are active on trastuzumab-resistant tumours

Background:

Overexpression of ErbB2 receptor in breast cancer is associated with disease progression and poor prognosis. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer, has proven to be effective; however, a relevant problem for clinical practice is that a high fraction of breast cancer patients shows primary or acquired resistance to trastuzumab treatment.

Methods:

We tested on trastuzumab-resistant cells two novel human anti-tumour immunoconjugates engineered in our laboratory by fusion of a human anti-ErbB2 scFv, termed Erbicin, with either a human RNase or the Fc region of a human IgG1. Both Erbicin-derived immunoagents (EDIAs) are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, target an ErbB2 epitope different from that recognised by trastuzumab and do not show cardiotoxic effects.

Results:

We report that EDIAs are active also on trastuzumab-resistant tumour cells both in vitro and in vivo, most likely because of the different epitope recognised, as EDIAs, unlike trastuzumab, were found to be able to inhibit the signalling pathway downstream of ErbB2.

Conclusion:

These results suggest that EDIAs are immunoagents that could not only fulfil the therapeutic need of patients ineligible to trastuzumab treatment due to cardiac dysfunction but also prove to be useful for breast cancer patients unresponsive to trastuzumab treatment.     

Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma

Background:

Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma.

Methods:

The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours.

Results:

Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis.

Conclusion:

The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.     

Frequency of HER2 expression of circulating tumour cells in patients with metastatic or recurrent gastrointestinal cancer

Background:

The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood.

Methods:

A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System.

Results:

Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours.

Conclusion:

The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.     

Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2-positive breast tumour recurrence

Background:

We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.

Methods:

We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.

Results:

Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.

Conclusion:

Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.     

Targeting ErbB3-mediated stromal-epithelial interactions in pancreatic ductal adenocarcinoma

Background:

We sought to investigate the role of ErbB3-mediated signalling on the interaction between pancreatic cancer-associated fibroblasts (CAF) and carcinoma cells in an effort to disrupt tumourigenic pancreatic ductal adenocarcinoma (PDAC) stromal–epithelial cross-communication.

Methods:

Primary CAF cultures were established from human PDAC surgical specimens. AsPC-1 pancreatic cancer cell murine subcutaneous xenografts were developed in the presence and absence of CAF and were subsequently treated with epidermal growth factor receptor (EGFR) inhibitors (erlotinib) and ErbB3 inhibitors (MM-121, monoclonal ErbB3 antibody).

Results:

Cancer-associated fibroblasts were found to secrete neuregulin-1 (NRG-1), which promoted proliferation via phosphorylation of ErbB3 and AKT in AsPC-1 PDAC cells. This signalling cascade was effectively inhibited both in vitro and in vivo by specific ErbB3 blockade with MM-121, with greater degree of tumourigenesis inhibition when combined with erlotinib. The CAF–AsPC-1 pancreatic cancer xenografts reached significantly greater tumour volume than those xenografts lacking CAF and were resistant to the anti-tumour effects of EGFR inhibition with erlotinib.

Conclusion:

Cancer-associated fibroblasts-derived NRG-1 promote PDAC tumourigenesis via ErbB3-AKT signalling and overcomes single-agent EGFR inhibition. Disruption of this stromally mediated tumourigenic mechanism is best obtained through combined EGFR-ErbB3 inhibition with both erlotinib and MM-121. We have identified the NRG-1/ErbB3 axis as an attractive molecular target for the interruption of tumourigenic stromal–epithelial interactions within the PDAC microenvironment.     

Exploring the use and impact of adjuvant Trastuzumab for HER2-positive breast cancer patients in a large UK cancer network Do the results of international clinical trials translate into a similar benefit for patients in South East Wales?

Background:

Trastuzumab was approved in the United Kingdom for adjuvant treatment of human epidermal growth factor receptor 2 (HER2)+ breast cancer in 2006 at significant economic cost and with limited evidence in smaller T1N0 tumours. The South East Wales Cancer Network covers a population of 1 420 000 and maintains a database of treatments used. We examined this database to ensure the outcome of Trastuzumab use is as expected, especially in patients with T1N0 cancers.

Ethods:

M Case notes of patients with HER2+ disease eligible for adjuvant Trastuzumab over 2005–2008 were reviewed. Disease-free survival (DFS) and overall survival (OS) were calculated with the Kaplan–Meier method using SPSS (version 16.0.01 for Windows, SPSS, Chicago, IL, USA).

Results:

A total of 239 of 338 (70.7%) eligible HER2+ patients received treatment. At 3 years, the DFS of the treated group was 90.3% vs 73.3% and the OS was 98.5% vs 87.6%. In all, 47 of 92 stage I patients received Trastuzumab. Despite a trend towards worse prognostic factors in the treated group the DFS was 100% vs 84.1% and the OS was 100% vs 93.3%.

Conclusion:

Our results are comparable to those from landmark Trastuzumab trials. As evidence continues to emerge that smaller HER2+ cancers may behave aggressively our analysis of stage I tumours adds further support to the use of Trastuzumab in these patients.     

Prospective neoadjuvant analysis of PET imaging and mechanisms of resistance to Trastuzumab shows role of HIF1 and autophagy

Background:

Although Trastuzumab has improved survival of HER2+ breast cancer patients, resistance to the agent pre-exists or develops through the course of therapy. Here we show that a specific metabolism and autophagy-related cancer cell phenotype relates to resistance of HER2+ breast cancer to Trastuzumab and chemotherapy.

Methods:

Twenty-eight patients with locally advanced primary breast cancer were prospectively scheduled to received one cycle of Trastuzumab followed by a new biopsy on day 21, followed by taxol/Trastuzumab chemotherapy for four cycles before surgery. FDG PET/CT scan was used to monitor tumour response. Tissue samples were immunohistochemically analysed for metabolism and autophagy markers.

Results:

In pre-Trastuzumab biopsies, the LC3A+/HER2+ cell population was correlated with HIF1α expression (P=0.01), while GLUT1 and LC3B expression were correlated with Ki67 proliferation index (P=0.01 and P=0.01, respectively). FDG PET tumour dimensions before therapy were correlated with LC3B expression (P=0.005). Administration of Trastuzumab significantly reduced clinical and PET-detected tumour dimensions (P<0.01). An inverse association of tumour response with the percentage of cells expressing HIF1α at baseline was documented (P=0.01). Administration of Trastuzumab resulted in a decrease of the proliferation index (P=0.004), GLUT1 (P=0.04) and HER2 (P=0.01) expression. In contrast, the percentage of LC3A+/HER2+ cells was increased (P=0.01). High baseline HIF1α expression was the only parameter associated with poorer pathological response to preoperative chemotherapy (P=0.001).

Conclusions:

As the HER2+/LC3A+ phenotype, which often overexpresses HIF1α, is a major subpopulation increasing after therapy with Trastuzumab, LC3A- and HIF1α-targeting therapies should be investigated for the augmentation of anti-HER2 therapy efficacy.     

Macrophage polarisation changes within the time between diagnostic biopsy and tumour resection in oral squamous cell carcinomas—an immunohistochemical study

Background:

The prognosis of solid malignancies has been shown to depend on immunological parameters, such as macrophage polarisation (M1/M2). Recently, it was reported that preoperative oral surgery leads to a worsening of oral squamous cell carcinomas (OSCC) prognosis. Diagnostic incision biopsies are oral surgery procedures that might lead to healing-associated M2 macrophage polarisation with a potential negative influence on tumour biology. No studies have compared macrophage polarisation in OSCC biopsies and tumour specimens.

Methods:

Preoperative diagnostic incision biopsies (n=25) and tumour resection specimens (n=34) of T1/T2 OSCC were processed for immunohistochemistry to detect CD68-, CD11c-, CD163- and MRC1-positive cells. Samples were digitised using whole-slide imaging, and the expression of macrophage markers was quantitatively analysed.

Results:

Carcinoma tissues obtained during OSCC tumour resections showed a significantly (P<0.05) increased CD163 cell count (M2 macrophages) compared with tissues obtained during preoperative incision biopsies. Additionally, the CD163/CD68 ratio (an indicator of M2 polarisation) was significantly (P<0.05) higher in tumour resection specimens than in biopsies.

Conclusions:

This study revealed for the first time an increase in M2 polarisation in samples obtained during OSCC tumour resection surgery compared with preoperative incision biopsies. The biopsy-induced tissue trauma might explain the observed shift in macrophage polarisation towards the tumour-promoting M2 type and could lead to accelerated tumour progression.     

Lapatinib monotherapy in patients with relapsed, advanced, or metastatic breast cancer: efficacy, safety, and biomarker results from Japanese patients phase II studies

Background:

HER2-positive metastatic breast cancer (MBC) relapsing after trastuzumab-based therapy may require continued HER2 receptor inhibition to control the disease and preserve the patients'' quality-of-life. Efficacy and safety of lapatinib monotherapy was evaluated in Japanese breast cancer patients after trastuzumab-based therapies.

Methods:

In studies, EGF100642 and EGF104911 evaluated the efficacy and safety of oral lapatinib given 1500 mg once daily in patients with advanced or MBC. All patients progressed on anthracyclines and taxanes; HER2-positive patients had also progressed on trastuzumab.

Results:

For HER2-positive tumours (n=100), objective response rate was 19.0% (95% confidence interval (CI): 11.8–28.1) and clinical benefit rate (CBR) was 25.0% (95% CI: 16.9–34.7). One out of 22 HER2-negative tumour was documented as complete response (n=22). The median time-to-progression (TTP) in the HER2-positive and HER2-negative groups was 13.0 and 8.0 weeks (P=0.007); median overall survival was 58.3 and 40.0 weeks, respectively. The most frequent adverse event was diarrhoea. TTP and CBR were significantly associated with HER2 expression. Patients with tumours harbouring an H1047R PIK3CA mutation or low expression of PTEN derived clinical benefit from lapatinib.

Conclusion:

Lapatinib monotherapy had shown anti-tumour activity in Japanese patients with HER2-positive MBC that relapsed after trastuzumab-based therapy, including those with brain metastases. Patients benefiting from lapatinib may have biomarker profiles differing from that reported for trastuzumab.     

Pathological complete response and prognosis after neoadjuvant chemotherapy for HER2-positive breast cancers before and after trastuzumab era: results from a real-life cohort

Background:

Trastuzumab was introduced a decade ago and has improved outcomes for HER2-positive breast cancer. We investigated the factors predictive of pathological complete response (pCR), prognostic factors for disease-free survival (DFS), and interactions between pCR and DFS after neoadjuvant treatment.

Methods:

We identified 287 patients with primary HER2-positive breast cancers given neoadjuvant chemotherapy (NAC) between 2002 and 2011. Univariate and multivariate analyses of clinical and pathological factors associated with pCR and DFS were performed.

Results:

pCR rates differed between patients receiving neoadjuvant trastuzumab treatment or not (47.7% versus 19.3%, P<0.0001). DFS also differed significantly between patients receiving adjuvant trastuzumab or not (hazard ratio=4.84, 95% CI (2.52; 9.31), P<0.001). We analysed 199 patients given neoadjuvant and adjuvant trastuzumab. Multivariate analysis identified older age and hormone receptor-negative tumours as independent predictors of pCR. T stage (hazard ratio=2.55, 95% CI (1.01; 6.48), P=0.05) and strict pCR (hazard ratio=9.15, 95% CI (1.22; 68.83), P=0.03) were independent predictors of DFS. The latter association was significant in the HR-negative subgroup (P=0.02) but not in the HR-positive subgroup (P=0.12).

Conclusions:

Major pCR and DFS gains in HER2-positive BC were observed since ‘trastuzumab'' era. Further improvements rely on the enrollment of accurately selected patients into clinical trials.     

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:

Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.

Methods:

We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.

Results:

The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.

Conclusions:

Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.     

Can shear-wave elastography predict response to neoadjuvant chemotherapy in women with invasive breast cancer?

Background:

Response of invasive breast cancer to neoadjuvant chemotherapy (NAC) is variable, and prediction of response is imperfect. We aimed to ascertain whether tissue stiffness in breast cancers, as assessed by shear-wave elastography (SWE) before treatment, is associated with response.

Methods:

We retrospectively compared pre-treatment tumour mean tissue stiffness, with post-treatment Residual Cancer Burden (RCB) scores and its components in 40 women with breast cancer treated by NAC using Pearson''s correlation coefficient (CC), a general linear model and multiple linear regression. Subgroup analysis was carried out for luminal, HER2-positive and basal immuno-histochemical subtypes.

Results:

Statistically significant correlations were shown between stiffness and RCB scores and between stiffness and percentage tumour cellularity. The correlation between stiffness and percentage cellularity was strongest (CC 0.35 (P<0.0001) compared with CC 0.23 (P=0.004) for the RCB score). The results of a general linear model show that cellularity and RCB score maintain independent relationships with stiffness. By multiple linear regression, only cellularity maintained a significant relationship with stiffness.

Conclusion:

Pre-treatment tumour stiffness measured by SWE, has a statistically significant relationship with pathological response of invasive breast cancer to NAC.     

Epithelial expression of FHL2 is negatively associated with metastasis-free and overall survival in colorectal cancer

Background:

Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to have a role in cancer progression. It has been shown to be overexpressed in the colorectal cancer (CRC).

Methods:

Here, we examined a possible prognostic value of FHL2 in CRC. Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at the time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using small-interfering RNA transfection in hTERT-immortalised myofibroblasts.

Results:

Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Multivariate Cox analysis shows that expression in the tumour invasion front (P<0.001) as well as in the centre of the tumour (P<0.001) was associated with metachronous metastases independently of the clinicopathological variables; expression in the tumour invasion front was also associated with overall survival independently of the clinicopathological variables (P<0.01).

Conclusion:

Higher FHL2 expression is involved in CRC progression and correlates with the development of metachronous metastases and overall survival, suggesting that FHL2 is an independent adverse prognostic indicator for CRC.     

Insulin-like growth factor I and risk of epithelial invasive ovarian cancer by tumour characteristics: results from the EPIC cohort

Background:

Prospective studies on insulin-like growth factor I (IGF-I) and epithelial ovarian cancer (EOC) risk are inconclusive. Data suggest risk associations vary by tumour characteristics.

Methods:

We conducted a nested case–control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate IGF-I concentrations and EOC risk by tumour characteristics (n=565 cases). Multivariable conditional logistic regression models were used to estimate associations.

Results:

We observed no association between IGF-I and EOC overall or by tumour characteristics.

Conclusions:

In the largest prospective study to date was no association between IGF-I and EOC risk. Pre-diagnostic serum IGF-I concentrations may not influence EOC risk.     

Immunotherapy augments the effect of 5-azacytidine on HPV16-associated tumours with different MHC class I-expression status

Background:

Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy.

Methods:

We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours.

Results:

We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8⁺-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed.

Conclusion:

Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours.     

Impact of anti-HER2 therapy on overall survival in HER2-overexpressing breast cancer patients with brain metastases

Background:

Trastuzumab-based therapy after diagnosis of brain metastases (BM) may improve survival due to prolonged systemic disease control. We investigated whether lapatinib may yield additional survival benefit.

Methods:

Eighty patients with BM from HER2-positive breast cancer were identified. Karnofsky Performance Score (KPS) of at least 70 was required. We included a control group of 37 patients treated before 2003, when continuation of trastuzumab after diagnosis of BM was not yet recommended. Remainders received either trastuzumab or lapatinib and trastuzumab (either concomitantly or sequentially) with or without chemotherapy.

Results:

Median overall survival (OS) in patients receiving trastuzumab after diagnosis of BM was 13 months; corresponding numbers were 9 months in patients treated with chemotherapy, and 3 months with radiotherapy alone. Median OS was not reached in the lapatinib group. Addition of lapatinib prolonged OS over trastuzumab alone (P=0.002). After correction for potential confounders, lapatinib therapy remained an independent positive predictor for survival (HR 0.279; P=0.012).

Interpretation:

This retrospective single-centre study suggests that the introduction of lapatinib improved survival in patients with BM from HER2-positive breast cancer. Patients with KPS ⩾70 may benefit when treated with lapatinib in addition to trastuzumab after completion of local therapy.     

Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile

Background:

Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function.

Methods:

BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model.

Results:

Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance.

Conclusion:

BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.     

Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance

Background:

Human head and neck squamous cell carcinoma (HNSCC) fundamentally vary in their susceptibility to different cytotoxic drugs and treatment modalities. There is at present no clinically accepted test system to predict the most effective therapy for an individual patient.

Methods:

Therefore, we established tumour-derived slice cultures which can be kept in vitro for at least 6 days. Upon treatment with cisplatin, docetaxel and cetuximab, slices were fixed and paraffin sections were cut for histopathological analysis.

Results:

Apoptotic fragmentation, activation of caspase 3, and cell loss were observed in treated tumour slices. Counts of nuclei per field in untreated compared with treated slices deriving from the same tumour allowed estimation of the anti-neoplastic activity of individual drugs on an individual tumour.

Conclusion:

HNSCC-derived slice cultures survive well in vitro and may serve not only to improve personalised therapies but also to detect mechanisms of tumour resistance by harvesting surviving tumour cells after treatment.     

Regulation of tamoxifen sensitivity by a PAK1–EBP1 signalling pathway in breast cancer

Background:

EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells.

Methods:

Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay.

Results:

Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells.

Conclusion:

These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed.     

In vitro and in vivo inhibitory effect of three Cox-2 inhibitors and epithelial-to-mesenchymal transition in human bladder cancer cell lines

Background:

Although the anti-tumour effect of cyclooxygenase-2 (Cox-2) inhibitors in invasive bladder cancer has been confirmed, its mechanisms of action are unclear. Recently, the concept of an epithelial-to-mesenchymal transition (EMT) promoting carcinoma progression has been suggested, and a key feature of the EMT is the downregulation of E-cadherin. In this study, we investigated the effect of Cox-2 inhibitors on reversal EMT and tumour growth inhibition in bladder cancer cells.

Methods:

We used three Cox-2 inhibitors, etodolac, celecoxib and NS-398 and three human bladder cancer cell lines, T24, 5637 and KK47, in this study. T24 xenograft tumour mouse model was used in the in vivo study.

Results:

Within the clinical drug concentrations, only etodolac showed the in vitro growth inhibition in T24 not in the other cell lines. Etodolac reduced SNAIL mRNA and vimentin cell surface expression, and induced E-cadherin mRNA and E-cadherin cell surface expression, in T24. Etodolac also most strongly inhibited the cell migration of T24 in vitro and showed the highest tumour growth inhibition in T24 tumour in vivo.

Conclusion:

Etodolac at clinical doses exhibited induced in vitro and in vivo anti-tumour effects and reversal effect of EMT in T24. These results suggest that etodolac is a good candidate for an anti-tumour or chemopreventive reagent for high-grade bladder cancer.