Trastuzumab in the adjuvant treatment of HER2-positive early breast cancer patients: a meta-analysis of published randomized controlled trials

Background

Adjuvant trastuzumab therapy has yielded conflicting results for overall survival, concerns about central nervous system (CNS) metastasis, and questions about optimal schedule. Therefore, we carried out a meta-analysis to assess the benefits of concurrent or sequential trastuzumab with adjuvant chemotherapy for early breast cancer patients with HER2-positive tumors.

Methods

Computerized and manual searches were performed to identify randomized clinical trials comparing adjuvant chemotherapy with or without trastuzumab in HER2-positive early breast cancer patients. Odds ratios were used to estimate the association between the addition of trastuzumab to adjuvant chemotherapy and various survival outcomes. The fixed-effects or random-effects model was used to combine data.

Findings

With six eligible studies identified, this analysis demonstrated that patients with HER2-positive breast cancer derived benefit in disease-free survival, overall survival, locoregional recurrence and distant recurrence (all P<0.001) from the addition of trastuzumab to adjuvant chemotherapy, whereas trastuzumab did worse in CNS recurrence as compared to the control group (P = 0.018). Furthermore, concomitant use of trastuzumab significantly lowered the hazard of death (P<0.001) but bore a higher incidence of CNS recurrence (P = 0.010), while statistical significance failed to be discerned for either overall survival (P = 0.069) or CNS metastasis (P = 0.374) between the sequential and observation arms.

Conclusion

This analysis verifies the efficacy of trastuzumab in the adjuvant setting. Additionally, our findings indirectly corroborate the superiority of concurrent trastuzumab to sequential use and also illuminate that prolonged survival is the possible reason for the higher incidence of CNS with trastuzumab versus observation.     

Darpp-32 and Its Truncated Variant t-Darpp Have Antagonistic Effects on Breast Cancer Cell Growth and Herceptin Resistance

Background

Herceptin (trastuzumab) is a humanized monoclonal antibody that is approved for the treatment of metastatic breast cancer patients whose tumors overexpress Her2 (erbB2/neu). Up to 70% of Her2-positive breast cancers demonstrate a response to Herceptin-based therapies, but resistance almost inevitably arises within a year of the initial response. To help understand the mechanism of Herceptin resistance, we isolated clonal variants of Her2-positive BT474 human breast cancer cells (BT/Her^R) that are highly resistant to Herceptin. These cell lines exhibit sustained PI3K/Akt signaling as an essential component of Herceptin-resistant proliferation. Several genes in the protein kinase A (PKA) signaling network have altered expression in BT/Her^R cells, including PPP1R1B, which encodes a 32 kDa protein known as Darpp-32 and its amino-terminal truncated variant, t-Darpp. The purpose of the current work was to determine the role of Darpp-32 and t-Darpp in Herceptin resistance.

Methodology and Results

We determined expression of Darpp-32 and t-Darpp in BT/Her^R cells selected for resistance to Herceptin. Subsequently, cDNAs encoding the two isoforms of Darpp-32 were transfected, separately and together, into Her2-positive SK-Br-3 breast cancer cells. Transfected cells were tested for resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. DNA binding activity by the cAMP response element binding protein (CREB) was also measured. We found that BT/Her^R cells overexpressed t-Darpp but not Darpp-32. Moreover, t-Darpp overexpression in SK-Br-3 cells was sufficient for conferring resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. Darpp-32 co-expression reversed t-Darpp''s effects on Herceptin resistance and Akt phosphorylation. t-Darpp overexpression led to increased CREB binding activity, which was also reversible by Darpp-32.

Conclusions

t-Darpp and Darpp-32 appear to have antagonistic effects on Herceptin resistance. We present a unified model by which these effects might be mediated via the PKA regulatory network.     

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Background

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors.

Methods and Findings

As proof-of-concept, we selected Herceptin™ (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice.

Conclusions

Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.     

Sprouty 2 is an independent prognostic factor in breast cancer and may be useful in stratifying patients for trastuzumab therapy

Background

Resistance to trastuzumab is a clinical problem, partly due to overriding activation of MAPK/PI3K signalling. Sprouty-family proteins are negative regulators of MAPK/PI3K signalling, but their role in HER2-therapy resistance is unknown.

Patients and Methods

Associations between Sprouty gene expression and clinicopathological features were investigated in a breast cancer microarray meta-analysis. Changes in expression of Spry2 and feedback inhibition on trastuzumab resistance were studied in SKBr3 and BT474 breast carcinoma cell lines using cell viability assays. Spry2 protein expression was measured by quantitative immunofluorescence in a cohort of 122 patients treated with trastuzumab.

Results

Low gene expression of Spry2 was associated with increased pathological grade, high HER2 expression, and was a significant independent prognostic factor. Overexpression of Spry2 in SKBr3s resulted in enhanced inhibition of cell viability after trastuzumab treatment, and the PI3K-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 had a similar effect. Low Spry2 expression was associated with increased risk of death (HR = 2.28, 95% CI 1.22–4.26; p = 0.008) in trastuzumab-treated patients, including in multivariate analysis. Stratification of trastuzumab-treated patients using PTEN and Spry2 was superior to either marker in isolation.

Conclusion

In breast cancers with deficient feedback inhibition, combinatorial therapy with negative regulators of growth factor signalling may be an effective therapeutic strategy.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody suppress hepatic cancer cells in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.

Methodology/Principal Findings

We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.

Conclusions

Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.     

Mini Profile of Potential Anticancer Properties of Propofol

Background

Propofol (2, 6-diisopropylphenol) is an intravenous sedative-hypnotic agent administered to induce and maintain anesthesia. It has been recently revealed that propofol has anticancer properties including direct and indirect suppression of the viability and proliferation of cancer cells by promoting apoptosis in some cancer cell lines.

Methodology/Principal Findings

This study aimed to establish a profile to quantitatively and functionally evaluate the anticancer properties of propofol in three cancer cell lines: non-small cell lung carcinoma cell line A549, human colon carcinoma cell line LoVo, and human breast cancer cell line SK-BR-3. We demonstrated that the expression level of caspase-3, an apoptosis biomarker, significantly increased in a dose-dependent manner after 24-h stimulation with 100 µM propofol in A549 cells, and slightly increased in LoVo cells. However, there was no change in caspase-3 expression in SK-BR-3 cells. High caspase-3 expression in A549 cells may be modulated by the ERK1/2 pathway because phosphorylated ERK1/2 dramatically reduced after propofol treatment. BAX, a major protein that promotes apoptosis in the regulation phase, was highly expressed in A549 cells after treatment with 25 µM propofol. Apoptosis induced by propofol may be associated with cancer cells carrying Kras mutations.

Conclusions/Significance

Our results suggest that the anti-cancer effects of propofol, which are consistent with those of previous studies, are likely associated with the Kras mutation status. Only Kras mutation in Codon 12 instead of other Kras status has been demonstrated to play an important role in sensitizing the propofol-induced apoptosis in cancer cell lines from our study. These findings may enable us a detailed investigation of propofol/Kras-mediated cancer cell apoptosis in the future.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

The role of individual caspases in cell death induction by taxanes in breast cancer cells

Background

In previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3).

Methods and results

Using western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment.

Conclusion

We conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.     

Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody

Background

The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents.

Methods

Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells.

Results

The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice.

Conclusions

A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.     

Assessment of Tracer 99mTc(V)-DMSA Uptake as a Measure of Tumor Cell Proliferation In Vitro

Purpose

To examine whether ^99mTc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation.

Methods

Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with ^99mTc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-³H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.

Results

In all cell lines rates of ^99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-³H]thymidine incorporation and expression of PCNA and Ki-67.

Conclusion

Rates of ^99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.     

Adult Stromal Cells Derived from Human Adipose Tissue Provoke Pancreatic Cancer Cell Death both In Vitro and In Vivo

Background

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.

Principal Findings

Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.

Conclusion

These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.     

Characterization of the PMT Gene Family in Cryptococcus neoformans

Background

Protein-O-mannosyltransferases (Pmt''s) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins.

Methodology/Principal Findings

Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis.

Conclusion/Significance

These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host.