Overexpressing Breast Cancer Cells Research Articles

P62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA

Aberrant p62 overexpression has been implicated in breast cancer development. Here, we found that p62 expression was elevated in breast cancer stem cells (BCSCs), including CD44+CD24− fractions, mammospheres, ALDH1+ populations and side population cells. Indeed, short-hairpin RNA (shRNA)-mediated knockdown of p62 impaired breast cancer cells from self-renewing under anchorage-independent conditions, whereas ectopic overexpression of p62 enhanced the self-renewal ability of breast cancer cells in vitro. Genetic depletion of p62 robustly inhibited tumor-initiating frequencies, as well as growth rates of BCSC-derived tumor xenografts in immunodeficient mice. Consistently, immunohistochemical analysis of clinical breast tumor tissues showed that high p62 expression levels were linked to poorer clinical outcome. Further gene expression profiling analysis revealed that p62 was positively correlated with MYC expression level, which mediated the function of p62 in promoting breast cancer stem-like properties. MYC mRNA level was reduced upon p62 deletion by siRNA and increased with p62 overexpression in breast cancer cells, suggesting that p62 positively regulated MYC mRNA. Interestingly, p62 did not transactivate MYC promoter. Instead, p62 delayed the degradation of MYC mRNA by repressing the expression of let-7a and let-7b, thus promoting MYC mRNA stabilization at the post-transcriptional level. Consistently, let-7a and let-7b mimics attenuated p62-mediated MYC mRNA stabilization. Together, these findings unveiled a previously unappreciated role of p62 in the regulation of BCSCs, assigning p62 as a promising therapeutic target for breast cancer treatments.

Pomolic acid inhibits metastasis of HER2 overexpressing breast cancer cells through inactivation of the ERK pathway

Expression of the CXC chemokine receptor-4 (CXCR4), a G protein-coupled receptor, and HER2, a receptor tyrosine kinase, strongly correlates with tumor progression and metastatic potential of breast cancer cells. We report the identification of pomolic acid (PA) as a novel regulator of HER2 and CXCR4 expression. We found that PA downregulated the expression of HER2 and CXCR4 in SKBR3 cells in a dose- and time-dependent manner. When investigated for the molecular mechanism(s), it was found that the downregulation of HER2 and CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation as indicated by downregulation of mRNA expression. Moreover, we show that PA inhibits phosphorylation of ERK and reduces NF-κB activation. Suppression of CXCR4 expression by PA correlated with the inhibition of CXCL12-induced invasion of HER2-overexpressing breast cancer cells. Overall, our results demonstrate for the first time that PA is a novel inhibitor of HER2 and CXCR4 expression via kinase pathways and may play a critical role in determining the metastatic potential of breast cancer cells.

A novel small-molecule inhibitor of 3-phosphoglycerate dehydrogenase

ABSTRACTSerine metabolism is likely to play a critical role in cancer cell growth. A recent study reports the identification of a novel small-molecule inhibitor of serine synthesis that targets 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine synthesis pathway, and selectively abrogates the proliferation of PHGDH overexpressing breast cancer cells.

Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells.

We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150–250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core.

Tumor-suppressing roles of miR-214 and miR-218 in breast cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are key post-transcriptional regulators of gene expression. MicroRNA-214 (miR-214) and microRNA-218 (miR-218) have shown the function of tumor suppressors in various types of human cancers. However, the biological functions of miR-214 and miR-218 in breast cancer have not been elucidated completely. The present study evaluated the expression and biological function of miR-214 and miR-218 in human breast cancer. Our results revealed that the expression of miR-214 and miR-218 were significantly decreased in breast cancer tissues compared with adjacent tissues. The aberrant expression of miR-214 and miR-218 were negatively associated with Ki-67, and the miR-218 expression was positively associated with progesterone receptor (PR) in breast cancer tissues. In vitro, the cell proliferation and migration were decreased, cell apoptosis was induced, and cell cycle was also disturbed in miR-214 or miR-218 overexpressed breast cancer cells. Our results demonstrated that miR-214 and miR-218 function as tumor suppressors in breast cancer, and may become biomarkers and potential therapeutic targets in breast cancer.

Abstract P5-04-15: Inhibitory effects of calcitriol and the vitamin D analogue paricalcitol in combination with chemotherapy on the growth of HER2 overexpressing breast cancer cells

Abstract Introduction: Despite recent advances in therapy, additional options for improving the outcomes of patients with HER2-overexpressing breast cancer (BC) are needed. The role of vitamin D in relation to BC outcomes is controversial. Both calcitriol and synthetic vitamin D analogues have been shown to increase sensitivity to chemotherapy in BC cell lines. In this study, we aimed to determine the effect of combined treatment with either calcitriol or the vitamin D analogue paricalcitol plus doxorubicin or paclitaxel in HER2+ BC cells. Methods: Cell characterization was performed using western blot for estrogen receptor (ER) α, progesterone receptor (PR) A-B, HER2 receptor and vitamin D receptor (VDR) A-B. Flow cytometry was used to determine the quantitative expression of HER2 in an established breast cancer cell line known to express VDR (SKBR3 [ERα-, PR A-B -, HER2+]). The effects of calcitriol and paricalcitol, alone or in combination with doxorubicin or paclitaxel, were evaluated using a Sulforhodamine B growth assay. We calculated IC20 inhibitory concentrations by non-linear regression analysis using sigmoidal fitting of dose-response curves. Results are presented as the mean cell proliferation percentage (%) ± standard deviation. Statistical analyses were carried out using one-way ANOVA and the Holm-Sidak method. Results: Calcitriol, paricalcitol, doxorubicin and paclitaxel inhibited cell proliferation in a dose dependent manner. Calcitriol 100nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 30.9% ± 24.1, compared to 74.6% ± 6.6 with calcitriol alone (p = 0.003) and 86.6% ± 4.1 with doxorubicin alone (p < 0.001). Similarly, the combination of calcitriol 100nM plus paclitaxel 0.001 μM inhibited the proliferation of SKBR3 cells to 11.6% ± 10.4, compared to 77.5% ± 6.1 with calcitriol alone (p < 0.001) and 50.7% ± 8.4 with paclitaxel alone (p < 0.001). Paricalcitol 25nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 49.1% ± 25.2, compared to 72.3 ± 13.2 with paricalcitol alone (p = 0.21) and with doxorubicin alone (p = 0.05). Paricalcitol 10nM in combination with paclitaxel 0.001μM inhibited the proliferation of SKBR3 cells to 26.1% ± 23.1, compared to 72.3% ± 13.2 with paricalcitol alone (p = 0.05) and 43.2% ± 12.1 with paclitaxel alone (p =0.285). Conclusions: Simultaneous treatment of SKBR3 cells with calcitriol plus either doxorubicin or paclitaxel showed a cooperative growth-inhibiting effect when compared with each cytotoxic drug alone. The combination of paricalcitol with each of the cytotoxic agents also showed a trend towards a higher growth-inhibiting effect, but failed to reach statistical significance when compared with each drug alone. Although these results point towards the presence of a cooperative antiproliferative effect of Vitamin D compounds on HER2+ BC cells when combined with chemotherapy, the concentrations needed to reach such an effect were high, which could potentially limit their use in clinical practice. Citation Format: Mateos-Soria AS, Garcia-Becerra RA, Diaz-Nieto L, Ventura-Gallegos JL, Jacobo-Herrera N, Soto-Perez-de-Celis E, Zentella-Dehesa A, Chávarri-Guera Y. Inhibitory effects of calcitriol and the vitamin D analogue paricalcitol in combination with chemotherapy on the growth of HER2 overexpressing breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-15.

Highly efficient nuclear delivery of anti-cancer drugs using a bio-functionalized reduced graphene oxide

Targeted drug delivery has become important, attractive and challenging in biomedical science and applications. Anti-HER2 antibody-conjugated poly-l-lysine functionalized reduced graphene oxide (anti-HER2-rGO-PLL) nanocarriers were prepared to efficiently deliver doxorubicin targeting at the nucleus of HER2 over-expressing cancer cells. The polycationic PLL was first covalently grafted to graphene oxide (GO) nanosheets followed by reduction to obtain rGO-PLL with high drug loading and good colloidal stability. The anti-HER2 antibodies were subsequently conjugated to the amino groups of PLL to achieve excellent cell uptake capability. Cellular uptake of anti-HER2-rGO-PLL into MCF7/HER2 cells is significantly higher than that of rGO-PLL due to the specific targeting of anti-HER2 to HER2 overexpressing breast cancer cells. Additionally the anti-HER2-rGO-PLL enables a fast accumulation of DOX inside the nucleus, its subcellular site of action. In vitro cytotoxicity measurements clearly reveal a seven fold improvement in the anticancer efficacy for anti-HER2-rGO-PLL/DOX in comparison to rGO-PLL/DOX. The enhanced anticancer efficacy could be ascribed to the different intracellular DOX distributions resulted from the different internalization routes that are energy-dependent macropinocytosis and energy-independent direct penetration by anti-HER2-rGO-PLL and rGO-PLL, respectively. The results demonstrate that anti-HER2 conjugated rGO-PLL developed is a promising vehicle for efficient nuclear delivery of chemotherapeutic agents to HER2 over-expressing tumours.

Sulfatase 2 promotes breast cancer progression through regulating some tumor-related factors.

In previous studies Sulf2 has been evidenced to play an important role in tumor progression through editing sulfate moieties on heparan sulfate proteoglycans (HSPGs) and modulating heparin binding growth factors. However, the role of Sulf2 in breast cancer progression is still poorly understood. In the present study, we hypothesized that Sulf2 promoted breast cancer progression. Two different breast cancer cell lines, MCF-7 and MDA-MB-231, were chosen for this study because of high and low Sulf2 expression levels. We also altered their Sulf2 expression by establishing Sulf2 knockdown and overexpressing breast cancer cell lines MCF-7 shSulf2 and MDA-MB-231 Sulf2. To evaluate the functions of Sulf2, cell proliferation, apoptosis, cell cycle, invasion, mobility and adhesion of these cell lines were measured in vitro, and xenograft formation, invasion and metastasis ability were examined in vivo. Furthermore, expression of related genes were screened and were certified in these cell lines. We found that Sulf2 increased breast cancer proliferation, invasion, mobility and adhesion both in vitro and in vivo. Sulf2 also decreased cisplatin inducing breast cancer apoptosis without affecting the cell cycle. Sulf2 upregulated c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3) expression and downregulated the cluster of differentiation 82 (CD82) and platelet-derived growth factor C (PDGFC) expression in breast cancer. Our data confirmed that Sulf2 promoted breast cancer progression and regulated the expression of tumor-related genes in breast cancer.

The effects of bufadienolides on HER2 overexpressing breast cancer cells.

HER2 is a proto-oncogene frequently amplified in human breast cancer, its overexpression is correlated with tamoxifen resistance and decreased recurrence-free survival. Arenobufagin and bufalin are homogeneous bufadienolides of cardiac glycosides agents. In this research, we studied the effects of arenobufagin and bufalin on cellular survival and proliferation of HER2 overexpressing breast cancer cells and the mechanism under the results including the direct effect on HER2 downstream pathways. Our results showed that arenobufagin and bufalin could significantly inhibit the proliferation and survival of HER2 overexpressing breast cancer cells, along with the declination of SRC-1, SRC-3, nuclear transcription factor E2F1, phosphorylated AKT, and ERK. And the combination of each bufadienolide in low dose with tamoxifen could significantly enhance the inhibitory effect of tamoxifen on HER2 overexpressing breast cancer cells. All above suggest that arenobufagin and bufalin may be potential therapy adjuvants for HER2 overexpressing breast cancer therapy.

Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2-Akt axis.

As breast cancer cells often develop chemoresistance, better therapeutic options are in search to circumvent it. Here we demonstrate that human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer cells resist docetaxel-induced cytotoxicity by upregulating HER-2 and its activity downstream, through Akt and mitogen-activated protein kinase (MAPK) pathways. We observed that introducing resveratrol as a chemosensitizer in docetaxel chemotherapy blocks upregulation and activation of HER-2 in addition to blocking downstream signaling pathways such as Akt. Resveratrol and docetaxel combination results in the synergistic induction of cell death in HER-2-overexpressing SK-BR-3 cells, whereas introduction of wild-type HER-2 in MDA-MD-231 cells increased the resistance to docetaxel. Dominant-negative HER-2 sensitizes SK-BR-3 cells to docetaxel. Our study identified a new synergistic therapeutic combination that targets HER-2-induced breast cancer resistance and might help to overcome therapeutic resistance during breast cancer therapy. The synergism of docetaxel and resveratrol was maximum in SK-BR-3, which is unique among the cell lines studied, due to its high expression status of HER-2, a receptor known to dictate the signaling environment of breast cancer cells. Docetaxel could further induce HER-2 activity in these cells, which was downregulated on resveratrol treatment. Transfection of DN-HER-2 in SK-BR-3 cells inhibits the synergism as the transfection itself sensitizes these cells to docetaxel, leaving no role for resveratrol, whereas ectopic expression of HER-2 introduces the synergism in MDA-MB-231, the triple-negative cell line, in which the synergism was minimum, attesting the crucial role of HER-2 in suppressing the sensitivity to docetaxel. Single-agent docetaxel induced HER-2-mediated resistance to cell death, which was blocked by resveratrol. Resveratrol also downregulated docetaxel-induced activation of MAPK and Akt, survival signaling pathways downstream of HER-2. In short, this study, for the first time, establishes the role of HER-2–Akt signaling axis in regulating the synergistic effect of docetaxel and resveratrol in breast cancer cells overexpressing HER-2.

Synthesis of folate- pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing breast cancer cells.

The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)-functionalized poly(ethylene glycol)-poly(propylene succinate) (PEG-PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by (1)H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC-UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5%) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA-PEG-PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG-PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA-PEG-PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor.

Construction of plvx-cyclooxygenase-2-DsRed vector and its effects on proliferation in cyclooxygenase-2 overexpressed breast cancer cell line

Objective To construct plvx-cyclooxygenase-2(COX-2)-DsRed and establish breast cancer cell line MCF7 which overexpressed COX-2, to explore the effect of COX-2 on breast cancer cell. Methods The full-length COX-2 PCR product was obtained by total COX-2 PCR primers and COX-2 cDNA vector. After the PCR product and lentiviral vector plvx-DsRed-Monomer-N1 were cut simultaneously by restriction enzyme BamH1 and Xhol1, they were connected and sequenced, to get lentiviral vector plvx-COX-2-DsRed. After selected by puromycin, overexpressed COX-2 breast cancer cell line MCF7-plvx-COX-2-DsRed was obtained. The stable cell line was verified by real time PCR and Western blot. The differences of proliferation ability between stable cell line and normal one were compared by colony formation test and Western blot. Results The lentiviral vector plvx-COX-2-DsRed and stable cell line MCF7-plvx-COX-2-DsRed after selecting were obtained. COX-2 expression level of the stable cell line was 75.29 times as high as that of MCF7, and 64.91 times as high as that of cell line MCF7-plvx-DsRed-Monomer-N1 by PCR assay (P< 0.05), which was consistent with the results of Western blot and microscope photo. MTT results showed that cell line MCF7-plvx-COX-2-DsRed had grown faster than cell line MCF7 and MCF7-plvx-DsRed-Monomer-N1 from the 2nd day (P< 0.05), which was accordant with colony formation assay. MCF7-plvx-COX-2-DsRed cell line had higher c-myc expression and lower β-catenin expression than MCF7 cell and cell line MCF7-plvx-DsRed-Monomer-N1 detected by Western blot relative quantification (P< 0.05). Conclusion The plvx-COX-2-DsRed lentiviral vector and cell line MCF7-plvx-COX-2-DsRed are successfully constructed. COX-2 can increase proliferation of MCF7 cells through up-regulating the expression of c-myc. Key words: Cyclooxygenase; Breast neoplasms; Cell line; Lentivirus; Cell proliferation

Apoptotic effect of tannic acid on fatty acid synthase over-expressed human breast cancer cells

Breast cancer is one of the most common cancers and is the second leading cause of cancer mortality in women worldwide. Novel therapies and chemo-therapeutic drugs are urgently needed to be developed for the treatment of breast cancer. Increasing evidence suggests that fatty acid synthase (FAS) plays an important role in breast cancer, for the expression of FAS is significantly higher in human breast cancer cells than in normal cells. Tannic acid (TA), a natural polyphenol, possesses significant biological functions, including bacteriostasis, hemostasis, and anti-oxidant. Our previous studies demonstrated that TA is a natural FAS inhibitor whose inhibitory activity is stronger than that of classical FAS inhibitors, such as C75 and cerulenin. This study further assessed the effect and therapeutic potential of TA on FAS over-expressed breast cancer cells, and as a result, TA had been proven to possess the functions of inhibiting intracellular FAS activity, down-regulating FAS expression in human breast cancer MDA-MB-231 and MCF-7 cells, and inducing cancer cell apoptosis. Since high-expressed FAS is recognized as a molecular marker for breast cancer and plays an important role in cancer prognosis, these findings suggest that TA is a potential drug candidate for treatment of breast cancer.

Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2.

BackgroundTrastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model.ResultsTrastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are potential substrates of SHP-1.ConclusionTaken together, we have demonstrated that the SHP-1 is a negative regulatory factor of the tyrosine kinase activity of HER-2 and EGFR through inhibiting phosphorylation. Dual targeting of EGFR and HER-2, by combining trastuzumab with SHP-1 overexpression, may improve response in HER-2 overexpressing breast cancer cells that also express high levels of EGFR.

Tumor inhibitory effects of 131I-Trastuzumab on human epidermal growth factor receptor 2 overexpressing breast cancer cells and its possible mechanisms

Objective To explore antitumor effect of 131I-Trastuzumab on human epidermal growth factor receptor(HER)2 overexpressing breast cancer cells and investigate its possible mechanism. Methods The expression levels of HER2 of three different breast cancer cell lines (BT474, MCF-7, HCC1937) were detected with immunofluorescence. Trastuzumab was labeled with 131I using the Iodogen method and 131I-Trastuzumab was isolated with ultrafiltration membrane, then the labeling efficiency, radiochemical purity and immunoreactivity were measured. The effects of 131I, Trastuzumab and 131I-Trastuzumab on viability of BT474 cells were evaluated with cell counting kit-8 (CCK-8) assay. The levels of total Akt and phosphorylated Akt (p-Akt) were detected with Western blot analysis. One-way analysis of variance (ANOVA), ANOVA for factorial design, Bonferroni correction and Pearson correlation analysis were used for data analysis. Results The expression level of HER2 in BT474 cells was much higher than those in HCC1937 and MCF-7 cells. The labeling efficiency, radiochemical purity and immunoreactivity of 131I-Trastuzumab were (89.71±2.93)%, (91.80±1.43)% and (58.84±3.35)% respectively. 131I (4.625 GBq/L), Trastuzumab(125.0 mg/L) and 131I-Trastuzumab(4.625 GBq/L) exhibited a dose-dependent cytotoxicity against BT474 cells (r=-0.964, -0.912, -0.618; all P 0.05). P-Akt expression in both Trastuzumab group and 131I-Trastuzumab group were much lower than those of control group and 131I group (t=12.524, 15.984, 7.347, 10.807; all P 0.05). Conclusions 131I-Trastuzumab may kill HER2 overexpressing breast cancer cells more effectively than Trastuzumab alone. The underlying mechanism may be attributed to that 131I-Trastuzumab may enhance the radiosensitivity by the inhibitory effect on PI3K/Akt pathway and thus exert synergistic effects with 131I. Key words: Breast neoplasms; Receptors, epidermal growth factor-urogastrone; Antibodies, monoclonal; Iodine radioisotopes; Tumor cells, cultured

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 4188: Conditional epithelial cell-specific knockout of CCN6/Wisp3 disrupts normal development of the virgin murine mammary gland

Abstract Background: Breast cancer remains one of the top causes of morbidity and mortality among cancer patients in the U.S. Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. Our laboratory has shown that CCN6/Wisp3, a secreted extracellular matrix-associated protein which belongs to the CCN family, is lost in 80% of IBC compared to stage-matched non-IBC tumors. We have shown previously that CCN6 knockdown in benign human breast cells leads to an epithelial-to-mesenchymal transition and increased resistance to anoikis. Further, CCN6 overexpression in breast cancer cells reduces invasion and growth in vivo and in vitro. However, the consequences of CCN6 knockout in the mammary gland have never been explored. We hypothesized that the conditional deletion of CCN6 in mammary epithelial cells in mice may lead to defects in mammary gland development and may induce mammary tumors. Methods: We created a novel transgenic mouse model in which CCN6 is conditionally knocked out in mammary epithelial cells using the Cre-Lox recombination system. We collected groups of CCN6 knockout mice and littermate controls at 8 weeks, 4 months, and 12 months of age to observe the effect of CCN6 knockout on the development of the virgin mammary gland and on de novo tumor formation in aged mice. We also collected groups of CCN6 knockout and control mice at early pregnancy, late pregnancy, lactation and involution timepoints. Mammary whole mounts were prepared in order to characterize the phenotype, and portions of the inguinal gland were collected for immunohistochemical, protein and RNA analysis. Results: Conditional CCN6 knockout in the murine mammary gland results in defective mammary gland development in pubertal (8-week-old) and post-pubertal (4-month-old) virgin female mice compared to controls. At 8 weeks of age, virgin CCN6 knockout mice exhibit significantly fewer terminal end buds and fewer bifurcated terminal end buds. At 4 months of age, virgin CCN6 knockout mice have fewer lobuloalveolar units and a hypobranching phenotype compared to controls. We observed no phenotypic differences in the pregnancy and lactation timepoints between CCN6 knockout mice and controls, and no differences in pup weight or average litter size, suggesting that CCN6 is not critical for pregnancy and lactation. CCN6 knockout did not lead to spontaneous tumorigenesis in aged mice. Conclusion: We provide new evidence that CCN6 is an important signaling molecule in the mammary gland in vivo, as CCN6 is necessary for normal mammary development in virginal animals. We are currently analyzing the effects of CCN6 knockout in mammary gland involution after lactation, and the signaling pathways affected by CCN6 knockout in vivo. Further studies are needed to investigate effect of CCN6 knockout in breast cancer in vivo; to this end, we are currently crossing our CCN6 knockout line with the oncogenic MMTV-Pymt transgenic line. Citation Format: Emily E. Martin, Wei Huang, Jun-Lin Guan, Celina G. Kleer. Conditional epithelial cell-specific knockout of CCN6/Wisp3 disrupts normal development of the virgin murine mammary gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4188. doi:10.1158/1538-7445.AM2015-4188

Abstract 3557: Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib

Abstract Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3557. doi:10.1158/1538-7445.AM2015-3557

Abstract 134: CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer cells

Abstract Breast cancers are broadly classified into four different subtypes which are Luminal A, Luminal B, Basal type and Her2 positive cancers. The cells in Her2 positive tumor lesions overexpress the tyrosine kinase receptor ErbB2 or Her2 which serve as the major oncoprotein and drives the unrestrained proliferation of the cells which is one of the major hallmarks of cancer. About 25% of the breast cancer overexpress Her2 (ErbB2) proto-oncogene resulting in aggressive tumor phenotype and is associated in poor prognosis in patients. Though Her2 targeted therapies are being used hugely in clinical practice, a significant fraction of the Her2 positive tumors develop mechanisms to evade the targeted therapies of Her2 inhibition. CCN5 or Wisp2 is a matricellular protein which is now considered as an ‘anti-invasive gene’. It has been already that established from multiple evidences that CCN5 protein inhibits progression of triple negative breast cancer. The goal of the study is to investigate if CCN5 can regulate Her2 in Her2 positive breast cancer. We have found that CCN5 treatment also negatively regulates expression and activity of Her2 receptor in Her2 overexpressed breast cancer cells. It has been indicated from Her2 promoter assays that Wisp2/CCN5 negatively regulates the expression of Her2 in a transcriptional level. Wisp2 /CCN5 treatment of Her2 overexpressed breast cancer cell lines, SKBR3 and Bt474, exerts a negative impact on the migration and proliferative properties of the cancer cells. Thus it can be concluded that our initial studies on effect of CCN5 on Her2 positive cells can pave the way for a novel approach of therapy to control Her2 overexpressed tumor cell growth. Citation Format: Sandipto Sarkar, Gargi Maity, Amlan Das, Monami Majumder, Snigdha Banerjee, Sushanta Banerjee. CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2015-134

Abstract 2349: EGFR is transferred from triple negative breast cancer cells to immune cells via trogocytosis and expression of EGFR on immune cells is associated with high tumor grade of triple negative breast cancer patients

Abstract We and others previously reported that HER2 was transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis. In the current study, we report that EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without antibody administration. Interestingly, the EGFR-trogocytosed MDA-MB-231 cells reduced the expression of EGFR and changed their morphology to the spindle shape (well known as mesenchymal transition) and acquired greater cell motility. These cancer cell properties are representative as higher malignant potentials in breast cancer patients. Thus, we hypothesized that EGFR expression on tumor infiltrating lymphocytes (TILs), which probably is caused via EGFR trogocytosis by TILs, is correlated with higher tumor grade. We evaluated the EGFR expression of TILs in 36 triple negative breast cancer specimens and found the correlation between EGFR expression status on TILs and higher tumor grade (P = 0.02). These findings may explain the reason why triple negative breast cancer with greater proportion of TILs exhibit higher tumor grade and poor prognosis. Citation Format: Eiji Suzuki, Ayane Yamaguchi, Tatsuki R. Kataoka, Masahiro Hirata, Kosuke Kawaguchi, Mariko Nishie, Masakazu Toi. EGFR is transferred from triple negative breast cancer cells to immune cells via trogocytosis and expression of EGFR on immune cells is associated with high tumor grade of triple negative breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2015-2349