HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer.

SH2 domain proteins are important components of the signal transduction pathways activated by growth factor receptor tyrosine kinases. We have been cloning SH2 domain proteins by bacterial expression cloning using the tyrosine phosphorylated C-terminus of the epidermal growth factor receptor as a probe. One of these newly cloned SH2 domain proteins, GRB-7, was mapped on mouse chromosome 11 to a region which also contains the tyrosine kinase receptor, HER2/erbB-2. The analogous chromosomal locus in man is often amplified in human breast cancer leading to overexpression of HER2. We find that GRB-7 is amplified in concert with HER2 in several breast cancer cell lines and that GRB-7 is overexpressed in both cell lines and breast tumors. GRB-7, through its SH2 domain, binds tightly to HER2 such that a large fraction of the tyrosine phosphorylated HER2 in SKBR-3 cells is bound to GRB-7. GRB-7 can also bind tyrosine phosphorylated SHC, albeit at a lower affinity than GRB2 binds SHC. We also find that GRB-7 has a strong similarity over > 300 amino acids to a newly identified gene in Caenorhabditis elegans. This region of similarity, which lies outside the SH2 domain, also contains a pleckstrin homology domain. The presence of evolutionarily conserved domains indicates that GRB-7 is likely to perform a basic signaling function. The fact that GRB-7 and HER2 are both overexpressed and bound tightly together suggests that this basic signaling pathway is greatly amplified in certain breast cancers.     

Ramentaceone,a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells

The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer.     

S100A14, a Member of the EF-hand Calcium-binding Proteins,Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.     

Dynamic chromatin remodeling on the HER2 promoter in human breast cancer cells.

Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.     

Apigenin induces apoptosis through proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway

Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome.     

Dysregulation of HER2/HER3 signaling axis in Epstein-Barr virus-infected breast carcinoma cells

The role of Epstein-Barr virus (EBV) in the pathogenesis of breast cancer has been of long-standing interest to the field. Breast epithelial cells can be infected by EBV through direct contact with EBV-bearing lymphoblastoid cells, and EBV infection has recently been shown to confer breast cancer cells an increased resistance to chemotherapeutic drugs. In this study, we established EBV-infected breast cancer MCF7 and BT474 cells and demonstrated that EBV infection promotes tumorigenic activity of breast cancer cells. Firstly, we showed that the EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. The increased colony formation capacity in soft agar was associated with increased expression and activation of HER2/HER3 signaling cascades, as evidenced by the findings that the treatment of HER2 antibody trastuzumab (Herceptin), phosphatidylinositol 3-kinase inhibitor, or MEK inhibitor completely abolished the tumorigenic capacity. In the EBV-infected breast cancer cells, the expression of EBV latency genes including EBNA1, EBER1, and BARF0 was detected. We next showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 expression and promoted tumorigenic activity in MCF7 and BT474 cells by the use of both overexpression and small interfering RNA knock-down. Collectively, we demonstrated that EBV-encoded BARF0 promotes the tumorigenic activity of breast cancer cells through activation of HER2/HER3 signaling cascades.     

The mTOR Inhibitor Rapamycin Synergizes with a Fatty Acid Synthase Inhibitor to Induce Cytotoxicity in ER/HER2-Positive Breast Cancer Cells

Patients with ER/HER2-positive breast cancer have a poor prognosis and are less responsive to selective estrogen receptor modulators; this is presumably due to the crosstalk between ER and HER2. Fatty acid synthase (FASN) is essential for the survival and maintenance of the malignant phenotype of breast cancer cells. An intimate relationship exists between FASN, ER and HER2. We hypothesized that FASN may be the downstream effector underlying ER/HER2 crosstalk through the PI3K/AKT/mTOR pathway in ER/HER2-positive breast cancer. The present study implicated the PI3K/AKT/mTOR pathway in the regulation of FASN expression in ER/HER2-positive breast cancer cells and demonstrated that rapamycin, an mTOR inhibitor, inhibited FASN expression. Cerulenin, a FASN inhibitor, synergized with rapamycin to induce apoptosis and inhibit cell migration and tumorigenesis in ER/HER2-positive breast cancer cells. Our findings suggest that inhibiting the mTOR-FASN axis is a promising new strategy for treating ER/HER2-positive breast cancer.     

Phosphoproteomic screen identifies potential therapeutic targets in melanoma

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.     

Molecular Biomarker Analyses Using Circulating Tumor Cells

Background

Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.

Methodology/Principal Findings

Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.

Conclusions/Significance

Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.     

PRP4K is a HER2-regulated modifier of taxane sensitivity

The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified in vitro, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.     

Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR,HER2 and HER3

The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.     

HER2 Phosphorylates and Destabilizes Pro-Apoptotic PUMA,Leading to Antagonized Apoptosis in Cancer Cells

HER2 is overexpressed in 15–20% of breast cancers. HER2 overexpression is known to reduce apoptosis but the underlying mechanisms for this association remain unclear. To elucidate the mechanisms for HER2-mediated survival, we investigated the relationship between HER2 and p53 upregulated modulator of apoptosis (PUMA), a potent apoptosis inducer. Our results showed that HER2 interacts with PUMA, which was independent of HER2 activation. In addition, we observed that HER2 interacted with PUMA in both mitochondrial and non-mitochondrial compartments. We next examined whether HER2 phosphorylates PUMA. Notably, PUMA tyrosine phosphorylation has never been reported. Using an intracellular assay, we found PUMA to be phosphorylated in breast cancer cells with activated HER2. Via cell-free HER2 kinase assay, we observed that PUMA was directly phosphorylated by HER2. Activation of HER2 decreased PUMA protein half-life. To identify which of the three tyrosines within PUMA are targeted by HER2, we generated three PUMA non-phosphorylation mutants each with a single Tyr→Phe substitution. Results indicated that each PUMA single mutant had lost some, but not all phosphorylation by HER2 indicating that HER2 targets all three tyrosines. Consequently, we created an additional PUMA mutant with all three tyrosines mutated (TM-PUMA) that could not be phosphorylated by HER2. Importantly, TM-PUMA was found to have a longer half-life than PUMA. An inverse association was observed between HER2 and PUMA in 93 invasive breast carcinoma samples. We further found that TM-PUMA suppressed growth of breast cancer cells to a greater degree than PUMA. Also, TM-PUMA had a stronger propensity to induce apoptosis than PUMA. Together, our results demonstrate, for the first time, that PUMA can be tyrosine phosphorylated and that HER2-mediated phosphorylation destabilizes PUMA protein. The HER2-PUMA interplay represents a novel mechanism by which PUMA is regulated and a new molecular basis for HER2-mediated growth and survival of cancer cells.     

Structure-activity relationship of conformationally constrained peptidomimetics for antiproliferative activity in HER2-overexpressing breast cancer cell lines

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases and is involved in a signaling cascade for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating the role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (compound 5, Arg-[3-amino-3(1-napthyl)-propionic acid]-Phe) exhibited antiproliferative activity with IC(50) values in the nanomolar to micromolar range in breast cancer cell lines. To further investigate the structure-activity relationship of the compounds, various analogs of compound 5 were designed. Conformational constraints were initiated in the peptidomimetic with introduction of a Pro residue in the peptidomimetic sequence. Results of antiproliferative activity indicated that analogs of compound 5 with C-and N-terminal ends capped (compound 16) and compound 9 with Asp at the C-terminal exhibited antiproliferative activity in the lower micromolar range against breast cancer cell lines. Introduction of conformational constraints such as Pro residue in the sequence or cyclization did not enhance the activity of the peptidomimetic. Competitive binding studies were carried out to evaluate the binding of potent peptidomimetics to HER2-overexpressing cancer cell lines. Results indicated that compounds exhibiting antiproliferative activity in breast cancer cell lines bind to the cells that overexpress HER2 protein.     

Estrogen receptor alpha mediates epithelial to mesenchymal transition,expression of specific matrix effectors and functional properties of breast cancer cells

The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10 + cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10 + cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR–ERK signaling pathway.In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance.