Gene Expression Profiling Reveals Epithelial Mesenchymal Transition (EMT) Genes Can Selectively Differentiate Eribulin Sensitive Breast Cancer Cells

Objectives

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a mechanistically unique inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with eribulin activity compared to paclitaxel, which stabilizes microtubules, based on gene expression profiling of cell line panels of breast, endometrial, and ovarian cancer in vitro.

Results

We determined the sets of genes that were differentially altered between eribulin and paclitaxel treatment in breast, endometrial, and ovarian cancer cell line panels. Our unsupervised clustering analyses revealed that expression profiles of gene sets altered with treatments were correlated with the in vitro antiproliferative activities of the drugs. Several tubulin isotypes had significantly lower expression in cell lines treated with eribulin compared to paclitaxel. Pathway enrichment analyses of gene sets revealed that the common pathways altered between treatments in the 3 cancer panels were related to cytoskeleton remodeling and cell cycle regulation. The epithelial-mesenchymal transition (EMT) pathway was enriched in genes with significantly altered expression between the two drugs for breast and endometrial cancers, but not for ovarian cancer. Expression of genes from the EMT pathway correlated with eribulin sensitivity in breast cancer and with paclitaxel sensitivity in endometrial cancer. Alteration of expression profiles of EMT genes between sensitive and resistant cell lines allowed us to predict drug sensitivity for breast and endometrial cancers.

Conclusion

Gene expression analysis showed that gene sets that were altered between eribulin and paclitaxel correlated with drug in vitro antiproliferative activities in breast and endometrial cancer cell line panels. Among the panels, breast cancer provided the strongest differentiation between eribulin and paclitaxel sensitivities based on gene expression. In addition, EMT genes were predictive of eribulin sensitivity in the breast and endometrial cancer panels.     

Essential role for Rac in heregulin beta1 mitogenic signaling: a mechanism that involves epidermal growth factor receptor and is independent of ErbB4

Heregulins are a family of ligands for the ErbB3/ErbB4 receptors that play important roles in breast cancer cell proliferation and tumorigenesis. Limited information is available on the contribution of Rho GTPases to heregulin-mediated signaling. In breast cancer cells, heregulin beta1 (HRG) causes a strong activation of Rac; however, it does so with striking differences in kinetics compared to epidermal growth factor, which signals through ErbB1 (epidermal growth factor receptor [EGFR]). Using specific ErbB receptor inhibitors and depletion of receptors by RNA interference (RNAi), we established that, surprisingly, activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. Similar receptor requirements are observed for HRG-induced actin cytoskeleton reorganization and mitogenic activity via extracellular signal-regulated kinase (ERK). HRG-induced Rac activation was phosphatidylinositol 3-kinase dependent and Src independent. Furthermore, inactivation of Rac by expression of the Rac GTPase-activating protein beta2-chimerin inhibited HRG-induced ERK activation, mitogenicity, and migration in breast cancer cells. HRG mitogenic activity was also impaired by depletion of Rac1 using RNAi. Our studies established that Rac is a critical mediator of HRG mitogenic signaling in breast cancer cells and highlight additional levels of complexity for ErbB receptor coupling to downstream effectors that control aberrant proliferation and transformation.     

Design and synthesis of paclitaxel conjugated with an ErbB2-recognizing peptide, EC-1

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2. PTX is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer, yet patients have to suffer some side effects caused by its systemic toxicity. The aim of our conjugate is to specifically deliver antitumor agent PTX to breast cancer cells that overexpress oncogenic ErbB2 with the purpose to reduce toxicity and enhance selective killing of cancer cells. In this study, a concise and efficient synthetic route for the preparation of the PTX-EC-1 conjugate has been developed in 6% overall yield. This synthetic approach provides a general method for conjugating a highly functionalized and disulfide-bridge containing cyclopeptide to Taxol or other antitumor agents.     

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.     

PKCdelta and MAPK mediate G(1) arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G(1). Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCdelta inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCdelta, WTPKCdeltaAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCdeltaAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH(2)-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCdelta) in the G(1) arrest induced by PMA and suggested PKC as a target for breast cancer treatment.     

Unlocking Synergistic Potential: Agomelatine Enhances the Chemotherapeutic Effect of Paclitaxel in Breast Cancer Cell Through MT1 Melatonin Receptors and ER-alpha Axis

This study investigates the potential of agomelatine (AGO), a synthetic melatoninergic drug, in combination with paclitaxel (PTX) for the treatment of breast cancer. The effects of AGO, PTX and melatonin (MTN) on breast cancer cell viability were investigated, focusing on the role of MT1 receptors. Cell viability and gene expression were analyzed in MCF-7 and MDA-MB-231 breast cancer cell experiments. The results show that AGO has cytotoxic effects on breast cancer cells similar to MTN. Combining AGO and MTN with PTX showed synergistic effects in MCF-7 cells. The study also reveals differences in the molecular mechanisms of breast cancer between estrogen-positive MCF-7 cells and estrogen-negative MDA-MB-231 cells. Combination with AGO and PTX affects apoptosis-associated proteins in both cell types. The findings suggest that AGO, combined with PTX, may be a promising adjuvant therapy for breast cancer and highlight the importance of MTN receptors in its mechanism of action.     

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (~800 siRNAs, ~400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.     

Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3

Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy. However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs. It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors. In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance. High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance. To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3. A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance. Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone. Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs.     

Resveratrol suppresses the proliferation of breast cancer cells by inhibiting fatty acid synthase signaling pathway

In breast cancer cells, overexpression of human epidermal growth factor receptor 2 (HER2) increases the translation of fatty acid synthase (FASN) by altering the activity of PI3K/Akt signaling pathways. Cancer chemotherapy causes major side effects and is not effective enough in slowing down the progression of the disease. Earlier studies showed a role for resveratrol in the inhibition of FASN, but the molecular mechanisms of resveratrol-induced inhibition are not known. In the present study, we examined the novel mechanism of resveratrol on Her2-overexpressed breast cancer cells.The effect of resveratrol on the growth of breast cancer cells was assessed as percent cell viability by cytotoxicity-based MTT assay and the induction of apoptosis was determined by cell-death detection ELISA and flow cytometric analysis of Annexin-V–PI binding. Western immunobloting was used to detect signaling events in human breast cancer (SKBR-3) cells.Data showed that resveratrol-mediated down-regulation of FASN and HER2 genes synergistically induced apoptotic death in SKBR-3 cells. This concurrently caused a prominent up-regulation of PEA3, leads to down-regulation of HER2 genes. Resveratrol also alleviated the PI3K/Akt/mTOR signaling by down-regulation of Akt phosphorylation and up-regulation of PTEN expression.These findings suggest that resveratrol alters the cell cycle progression and induce cell death via FASN inhibition in HER2 positive breast cancer.     

ZD6474 enhances paclitaxel antiproliferative and apoptotic effects in breast carcinoma cells

Chemotherapy employing paclitaxel and docetaxel is widely used for treating early‐stage breast cancer and metastasis, which is frequently associated with overexpression of epidermal growth factor receptor (EGFR) and resistance to apoptosis. ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR, inhibits cell proliferation of solid tumors, including breast. Phase III clinical trials using ZD6474 in non‐small cell lung carcinoma when combined with standard chemotherapy appear promising. In order to improve the antineoplastic activity of paclitaxel, we presently investigated the effects of ZD6474 in combination with paclitaxel in EGFR and VEGFR expressing human breast cancer cell lines MCF‐7 and MDA‐MB‐231. ZD6474 synergistically decreased cell viability when used in combination with paclitaxel. ZD6474 inhibited cyclin D1 and cyclin E expression and induced p53 expression when combined with paclitaxel. The combination of ZD6474 with paclitaxel versus either agent alone also more potently down‐regulated the antiapoptotic bcl‐2 protein, up‐regulated pro‐apoptotic signaling events involving expression of bax, activation of caspase‐3 and caspase‐7 proteins, and induced poly(ADP‐ribose) polymerase resulting in apoptosis. ZD6474 combined with paclitaxel inhibited anchorage‐independent colony formation and invasion of breast cancer cells in vitro as compared to either single agent, indicating a potential involvement of altered expression and reorganization of cytoskeletal proteins in combinatorial treated breast cancer cells. Collectively, our studies indicate that incorporating an anti‐EGFR plus VEGFR strategy (ZD6474) with chemotherapy (paclitaxel), where clinical studies of dose‐intensive paclitaxel therapy are currently in progress, may be more effective in treating patients with locally advanced or metastatic breast cancer than either approach alone. J. Cell. Physiol. 226: 375–384, 2011. © 2010 Wiley‐Liss, Inc.     

Evaluation of synthetic isoflavones on cell proliferation, estrogen receptor binding affinity, and apoptosis in human breast cancer cells

Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.     

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.     

Methylseleninic acid enhances paclitaxel efficacy for the treatment of triple-negative breast cancer

A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.     

Synthesis and antitumor activity of peptide-paclitaxel conjugates.

Paclitaxel (Pac) is the most important anticancer drug used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. In this work, we present the synthesis of five 2'-paclitaxel-substituted analogs in which paclitaxel was covalently bound to peptides or as multiple copies to synthetic carriers. Ac-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Arg-NH(2), Folyl-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Ser-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](2)-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](3)-NH(2) and Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) were synthesized using 2'-halogeno-acetylated paclitaxel derivatives. Paclitaxel conjugates showed greater solubility in water than paclitaxel and inhibited the proliferation of human breast, prostate, and cervical cancer cell lines. Although all synthesized compounds had an antiproliferative activity, the Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) derivative showed improved biological activity in comparison with paclitaxel in cervical and prostate human cancer cells.     

Chemotherapy of breast cancer-additive anticancerogenic effects by 2-methoxyestradiol?

2-Methoxyestradiol (2ME) is an endogenous estradiol metabolite, which acts antiproliferative in various tumor cell lines independent of the hormone receptor status. We investigated whether combinations of 2ME with various chemotherapeutic or endocrine compounds may result in an additive effect on the proliferation of human breast cancer cells. The breast cancer cell lines MCF-7 (receptor-positive), BM (receptor-negative) and a MCF-7 line transfected with the aromatase gene were used. All cell lines were incubated in the concentration range of 0.8 microM to 25 microM with 2ME alone and in equimolar combinations with the following compounds: epirubicine, daunorubicine, paclitaxel, docetaxel, carboplatin, vinorelbine, 5-fluorouracil, mafosfamide and 4-OH tamoxifen. The effect of letrozole and 2ME alone and in equimolar combinations was tested in the concentration range of 0.6 to 1 microM. Proliferation was measured after 4 days using the ATP-chemosensitivity test. In MCF-7 cells 2ME in combination with 4OH-tamoxifen, epirubicine, docetaxel, 5-fluoprouracil, mafosfamide and carboplatin led to an additive effect. In BM cells only 2ME combined with 4OH-tamoxifen, daunorubicine and mafosfamide showed an additive action. Both letrozole and 2ME were nearly similar effective in inhibition of the aromatase gene. Here no additive effect was found. 2ME displayed antiproliferative actions in various human breast cancer cells. In addition 2ME was able to increase the antiproliferative property of certain antihormones and cytostatic substances. Furthermore 2ME exhibits a similar property as compared to letrozole in inhibiting the aromatase activity. Since 2ME was well tolerated in a recently conducted phase II trial in patients with refractory metastatic breast cancer, the combination of 2ME with chemotherapeutics or antihormones may offer a new clinically relevant treatment regimen.