Targeting the EGF receptor in breast cancer treatment

Summary Immunotoxins are a relatively new class of cytotoxic agents consisting of a catalytic toxin linked to an appropriate targeting ligand. The ligand directs the toxin to the surface of a tumor cell, whereupon the toxin enters the cell and catalytically inactivates the ribosome, thus disrupting protein synthesis and effecting cell death. Monoclonal antibodies (or their fragments) have been most commonly used to carry chemically conjugated toxins to proteins or antigens overexposed on the tumor cell surface, but specific ligands for tumor cell surface receptors could also provide effective targeting.The receptor for epidermal growth factor (EGFR) is overexpressed primarily in poor prognosis breast cancers that do not respond well to traditional therapies. Because EGFR is frequently overexpressed in breast cancer tissue and is associated with a poor prognosis, it is an attractive target for antitumor therapy.DAB ₃₈₉ EGF is an EGFR specific fusion toxin produced with recombinant DNA techniques consisting of sequences for the enzymatically active and membrane translocation domains of diphtheria toxin plus sequences for human epidermal growth factor. DAB ₃₈₉ EGF is a potent, EGFR specific, cytotoxic agent which rapidly inhibits protein synthesis by a mechanism of action similar to that of diphtheria itself. Preclinical studies in the laboratory and in animals now suggest the feasibility of investigating such an agent in the targeted therapy of patients with human breast cancer.     

Mechanisms of EGF receptor regulation in breast cancer cells

Overexpression of the EGF receptor in breast cancer correlates with poor prognosis and failure on endocrine therapy for both ER–/EGFR+ and ER+/EGFR+ tumors, suggesting a role for EGFR in the progression to hormone independence. The identification of specific DNase I hypersensitive site patterns for the EGFR gene in ER+ vs. ER– cells implicates regions of the EGFR first intron in up-regulation of EGFR, while estrogen regulation studies indicate the involvement of a repressor(s) in the maintenance of low levels of EGFR. Based on these findings, a multi-step model is proposed for the progression of breast cancer from a hormone-dependent, ER+/EGFR- phenotype to an aggressive, hormone-independent, ER–/EGFR+ stage.     

Ron receptor signaling augments mammary tumor formation and metastasis in a murine model of breast cancer

The tyrosine kinase receptor Ron has been implicated in several types of cancer, including overexpression in human breast cancer. This is the first report describing the effect of Ron signaling on tumorigenesis and metastasis in a mouse model of breast cancer. Mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK-/-) were crossed to mice expressing the polyoma virus middle T antigen (pMT) under the control of the mouse mammary tumor virus promoter. Both pMT-expressing wild-type control (pMT+/- TK+/+) and pMT+/- TK-/- mice developed mammary tumors and lung metastases. However, a significant decrease in mammary tumor initiation and growth was found in the pMT+/- TK-/- mice compared with controls. An examination of mammary tumors showed that there was a significant decrease in microvessel density, significantly decreased cellular proliferation, and a significant increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive staining in mammary tumor cells from the pMT+/- TK-/- mice compared with the pMT+/- TK+/+ mice. Biochemical analyses on mammary tumor lysates showed that whereas both the pMT-expressing TK+/+ and TK-/- tumors have increased Ron expression compared with normal mammary glands, the pMT-expressing TK-/- tumors have deficits in mitogen-activated protein kinase and AKT activation. These results indicate that Ron signaling synergizes with pMT signaling to induce mammary tumor formation, growth, and metastasis. This effect may be mediated in part through the regulation of angiogenesis and through proliferative and cell survival pathways regulated by mitogen-activated protein kinase and AKT.     

Fulvestrant regulates epidermal growth factor (EGF) family ligands to activate EGF receptor (EGFR) signaling in breast cancer cells

Estrogen receptor-α (ER) targeted therapies are routinely used to treat breast cancer. However, patient responses are limited by resistance to endocrine therapy. Breast cancer cells resistant to the pure steroidal ER antagonist fulvestrant (fulv) demonstrate increased activation of epidermal growth factor receptor (EGFR) family members and downstream ERK signaling. In this study, we investigated the effects of fulv on EGFR signaling and ligand regulation in several breast cancer cell lines. EGFR/HER2/HER3 phosphorylation and ERK1,2 activation were seen after 24–48 h after fulvestrant treatment in ER-positive breast cancer cell lines. 4-Hydroxy-tamoxifen and estradiol did not cause EGFR activation. Fulvestrant did not affect EGFR expression. Cycloheximide abolished the ability of fulv to activate EGFR suggesting the autocrine production of EGFR ligands might be responsible for fulvestrant induced EGFR signaling. qRT-PCR results showed fulv differentially regulated EGFR ligands; HB-EGF mRNA was increased, while amphiregulin and epiregulin mRNAs were decreased. Fulvestrant induced EGFR activation and upregulation of EGFR ligands were ER dependent since fulv treatment in C4-12, an ER-negative cell line derivative of MCF-7 cells, did not result in EGFR activation or change in ligand mRNA levels. ER downregulation by siRNA induced similar EGFR activation and regulation of EGFR ligands as fulvestrant. Neutralizing HB-EGF antibody blocked fulv-induced EGFR activation. Combination of fulv and EGFR family tyrosine kinase inhibitors (erlotinib and lapatinib) significantly decreased EGFR signaling and cell survival. In conclusion, fulvestrant-activated EGFR family members accompanied by ER dependent upregulation of HB-EGF within 48 h. EGF receptor or ligand inhibition might enhance or prolong the therapeutic effects of targeting ER by fulvestrant in breast cancer.     

Targeted therapies: last focus on EGF receptor inhibitors in breast cancer

EGF receptor is slightly expressed in breast cancer without any sufficiently impact on patient outcome. Nevertheless determination of EGFR level represents a certain interest in order to orientate a targeted therapy notably in the case of hormone therapy resistance. Based on preclinical findings, preliminary clinical study results confirms in vitro observations about the possibility to encompass the hormonotherapy or anti-HER2 resistance. But, future large clinical trials are necessary to redefine a new strategy to treat breast cancer patients.     

Leptin receptor expression and cell signaling in breast cancer

Obesity is considered a risk factor for many cancers, including breast cancer. Our laboratory has previously shown that leptin is mitogenic in many cancer cell lines, including breast. Information regarding the effects of high leptin levels on leptin receptor expression and signaling is lacking. The purpose of this study was to characterize leptin receptor expression in response to leptin in breast cancer cells. In addition, SOCS-3 expression (a leptin inducible inhibitor of leptin signaling), plus MAPK and PI3K signaling, were examined to determine their role in leptin-induced cell proliferation. Breast cancer cell lines, ZR75-1 and HTB-26, were treated with 0, 4, 40 or 80 ng/ml of leptin. Multiplex RT-PCR was performed to determine relative mRNA expression levels of the human short (huOB-Ra) or long (huOB-Rb) leptin receptor isoforms, or SOCS-3. MAPK and PI3K signaling was analyzed by phosphorylation of ERK and Akt, respectively, via Western blotting. Cell proliferation and inhibitor studies were analyzed by MTT assay. HTB-26 and ZR75-1 both expressed huOB-Ra, huOB-Rb and SOCS-3 mRNA; however, mRNA expression levels generally remained unchanged over time with leptin treatment. MAPK and PI3K pathways were activated in the presence of leptin over time. MAPK and PI3K inhibitors significantly blocked leptin-induced proliferation. Higher levels of circulating leptin contribute to breast cancer proliferation by activation of the MAPK and PI3K signaling pathways involved in cell growth and survival. The mitogenic effects of leptin are not a consequence of altered leptin receptor or SOCS-3 mRNA expression.     

Loss of heterozygosity of the oestrogen receptor gene in breast cancer.

DNA from 67 primary breast carcinoma biopsies has been examined for loss of heterozygosity (LOH) using the microsatellite (TA)n repeat marker positioned 1 kb upstream of the oestrogen receptor (ER) gene. Forty-seven (70.1%) of the cases were informative; nine of these (19.1%) were positive for LOH. In three of the nine cases, there was total loss, and in the other six cases there was a marked reduction in the intensity of signal from one allele. LOH correlated weakly with histological grade and age, but not with ER status. This result suggests that LOH of the ER gene does not have an important role in the lack of ER function in breast cancer tissues.     

Somatostatin receptor incidence and distribution in breast cancer using receptor autoradiography: relationship to EGF receptors

Somatostatin receptors (SS-R) were measured with in vitro receptor autoradiography using the SS analog 125I-[Tyr3]-SMS 201-995 as radioligand in 342 breast-tumor samples. In a group of 158 "small" tumor samples (mean section surface: 14 mm2 +/- 0.4; mean +/- SEM), 34 tumors (21%) were SS-R positive. In a group of 72 "large" tumor samples (mean size: 180 mm2 +/- 8; mean +/- SEM), 33 tumors (46%) were SS-R positive. In this second group, more than half of the tumors had a non-homogeneous distribution of SS-R, i.e., tumor regions within SS-R positive tumors were SS-R negative. In a group of 48 additional patients, we could show that primaries and their metastases, or double primaries from right and left breasts, or 2 primaries resected consecutively, could both occasionally be SS-R positive. Finally, in 71 SS-R-positive primary tumors, 18 tumor samples were found to have simultaneously Epidermal Growth Factor receptors (EGF-R); in 12 of these 18 cases, the 2 receptor types were not topographically overlapping. Whereas SS-R were located on tumor tissue, EGF-R were often seen on adjacent normal lobules and ducts. These results show that a subgroup of breast tumors contain SS-R, in several cases non-homogeneously distributed. Their location does not coincide with that of EGF-R. Metastasis of SS-R-positive primaries may be SS-R-positive, as are sometimes second primaries. For evaluation of SS-R incidence and distribution, autoradiography is of advantage, specially if it is performed on large tumor samples, since it allows precise identification of the tissue elements containing these receptors.     

Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research

Summary The EGF receptor (EGFR) and HER2 are members of a growth factor receptor family. Overexpression of either protein in advanced breast cancer correlates with poor prognosis. EGF stimulates growth by binding to EGFR, activating the receptor's intracellular tyrosine kinase. The initial consequence is phosphorylation of specific tyrosine-containing sequences in the receptor's carboxyl terminus. These phosphotyrosines serve as high affinity recognition sites for proteins that, in turn, transmit the growth signal inside the cell. Mechanistic studies suggest that EGF binds to a single EGFR, triggering dimerization with another like receptor molecule. This dimerization is thought to initiate the tyrosine kinase activation.The EGF receptor family was recently expanded with the sequencing of HER3 and HER4. Each of the four family members was postulated to regulate a unique growth or differentiation signaling repertoire when activated by a receptor-specific ligand. However, new data from numerous laboratories suggest that EGFR family members may play a complex and ultimately more flexible role in signaling by forming heterodimers between family members, e.g. EGFR:HER2 or HER4:HER2. These heterodimers may form even when only one member of the pair binds its ligand.This review summarizes current work on heterodimerization and attempts to predict the consequences for downstream signaling. In brief, when compared to ligand-dependent receptor homodimers comprised of two proteins with the same internalization sequence and phosphorylated tyrosine residues, heterodimers are likely to: i) expand substrate selection and downstream signaling pathway activation; ii) promote interaction between sets of substrates in the mixed receptor complexes that would not ordinarily be physically juxtaposed; iii) alter the duration of receptor signaling by changing rates of receptor internalization, ligand loss, kinase inactivation, recycling, etc.; and iv) alter rates of receptor and substrate dephosphorylation. In addition to understanding interactions of heterodimers with the internalization machinery, identification of receptor-specific substrates and binding proteins for each EGFR family member will be necessary to explicate the role of heterodimers in growth and differentiation.     

Arthritis augments breast cancer metastasis: role of mast cells and SCF/c-Kit signaling

Introduction

Breast cancer remains the second leading cause of cancer-related deaths for women in the United States. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We recently reported that mice with autoimmune arthritis have significantly increased incidence of bone and lung metastasis and decreased survival associated with breast cancer. In this study, we evaluated the mechanism underlying the increased metastasis.

Methods

We used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and another that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mast cell levels and metastasis were monitored.

Results

First, we confirmed that breast tumor-bearing arthritic mice have a significantly higher incidence of bone and lung metastasis than do their nonarthritic counterparts. Next, we showed increased recruitment of mast cells within the primary tumor of arthritic mice, which facilitates metastasis. Next, we report that arthritic mice without any tumors have higher numbers of mast cells in the bones and lungs, which may be the underlying cause for the enhanced lung and bone metastases observed in the arthritic mice. Next, we showed that once the tumor cells populate the metastatic niches (bones and lungs), they further increase the mast cell population within the niche and assist in enhancing metastasis. This may primarily be due to the interaction of c-Kit receptor present on mast cells and stem cell factor (SCF, the ligand for ckit) expressed on tumor cells. Finally, we showed that targeting the SCF/cKit interaction with an anti-ckit antibody reduces the differentiation of mast cells and consequently reduces metastasis.

Conclusion

This is the first report to show that mast cells may play a critical role in remodeling not only the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis through SCF/cKit interaction in breast cancer with arthritis.     

Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer

Earlier studies have demonstrated an unexplained depletion of the epidermal growth factor receptor (EGFR) protein expression in prostatic cancer. We now attribute this phenomenon to the presence of a variant EGFR (EGFRvIII) that is highly expressed in malignant prostatic neoplasms. In a retrospective study, normal, benign hyperplastic and malignant prostatic tissues were examined at the mRNA and protein levels for the presence of this mutant receptor. The results demonstrated that whilst EGFRvIII was not present in normal prostatic glands, the level of expression of this variant protein increased progressively with the gradual transformation of the tissues to the malignant phenotype. The selective association of high EGFRvIII levels with the cancer phenotype underlines the role that this mutant receptor may maintain in the initiation and progression of malignant prostatic growth, and opens the way for new approaches in the management of this disease including gene therapy.     

Folate binding protein and the estrogen receptor in breast cancer

Folate binding protein (FBP) and estrogen receptor (ER) content were determined in primary breast cancers of 48 patients. The mean FBP level was significantly higher in ER-negative tumors than in ER-positive tumors and largely independent of the degree of tumor involvement or menopausal status. FBP correlated negatively with ER and this was most marked for tumors from postmenopausal women. Since FBP may decrease available intracellular folate the present data support clinical findings that chemotherapeutic agents may be more effective for ER negative tumors.     

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.     

Role of estrogen receptor alpha in modulating IGF-I receptor signaling and function in breast cancer

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is a multifunctional transmembrane tyrosine kinase that has been implicated in neoplastic transformation. The tumorigenic potential of IGF-IR relies on its strong anti-apoptotic and mitogenic activity. The growth and survival signals of IGF-IR are mediated through multiple intracellular pathways, many of which emanate from insulin receptor substrate 1 (IRS-1). In hormone-dependent breast cancer cells, IGF-IR and IRS-1 are often co-expressed with the estrogen receptor alpha (ERalpha), and IGF-I and ER systems are engaged in a powerful functional cross-talk. Most notably, activation of ERalpha upregulates the expression of IRS-1, IGF-IR, and IGF-1, which results in amplification of IGF-I responses. Reciprocally, stimulation of IGF-IR increases the phosphorylation and activity of ERalpha. In contrast, in ERalpha-negative breast cancer cells and tumors, the levels of IGF-IR and IRS-1 are often decreased and IGF-I is non-mitogenic. Our data suggest that defective IGF-IR signaling in ERalpha-negative cells is related, at least in part, to improper activation of the IRS-1/PI-3K/Akt/GSK-3 pathway and lack of Rb1 phosphorylation. These defects are partially reversed by re-expression of ERalpha. Interestingly, some non-mitogenic IGF-I responses, such as migration and invasion are retained in the absence of ERalpha, suggesting that IGF-IR function in breast cancer cells might depend on the ERalpha status. The understanding of how ERalpha may dictate IGF-I responses will help in devising rational anti-IGF-IR strategies for breast cancer treatment.     

Mutant p53, EGF receptor and c-erbB-2 expression in human breast cancer.

p53 expression was studied in 111 primary breast cancers with a monoclonal antibody (PAb240) that reacts with an epitope in mutant p53. Epidermal growth factor receptors (EGFr) and oestrogen receptors (ER) measured by ligand binding, c-erbB-2 expression assessed by immunochemistry and lymph node status were compared with p53 staining. Fifty-nine tumours (53%) were positive for p53, and this correlated with EGFr expression (P less than 0.02), which is a known poor prognostic factor. Eighteen out of 59 p53+ tumours expressed c-erbB-2 versus 4 out of 52 p53- tumours assessed on paraffin sections. However, assessing c-erbB-2 by Southern blotting and immunochemistry on frozen sections showed that 20 out of 59 p53+ tumours overexpressed or had amplified c-erbB-2 compared with 15 out of 52 p53- tumours (not significant). Mean EGFr concentration in p53+ tumours was 31 fmol per mg of membrane protein versus 14 fmol mg-1 in p53- tumours. Cytoplasmic staining was the most frequent pattern found for p53, but some cases showed cytoplasmic plus nuclear staining, or focal cells with predominantly nuclear staining. Thus, abnormal p53 is the commonest oncogene abnormality described in breast cancer. The association with EGFr expression suggests that these oncogenes interact in the pathogenesis of breast cancer.     

The clinical relevance of unfolded protein response signaling in breast cancer

Protein homeostasis regulated by the Endoplasmic Reticulum (ER) is a recognized process involved in cancer progression. ER stress activates the Unfolded Protein Response (UPR) and has been implicated in a variety of cancers. Given the role of the UPR activation in carcinogenesis, we hypothesized that UPR activation could be associated with pathological progression, higher clinical stage, and worse survival in breast cancer. A total of 4,416 breast cancer patients from multiple independent cohorts were analyzed. We defined the UPR pathway score by the degree of enrichment by Gene Set Variant Analysis and median was used to divide high vs. low score groups in each cohort. High UPR breast cancer significantly enriched not only cell proliferation-related but also other pro-cancerous gene sets consistently in both METABIC and {"type":"entrez-geo","attrs":{"text":"GSE96058","term_id":"96058"}}GSE96058 cohort. Majority of UPR pathway score high cells in the bulk tumor were tumor cells compared to other cells, including stromal, T-, B-, and myeloid-cells (P<0.001). UPR score was significantly associated with advanced stage, high grade, and triple negative breast cancer (TNBC) (all P<0.001). High UPR breast cancer was associated with worse patient survival in both cohorts (all P<0.001). Among breast cancer subtype, ER-positive/HER2-negative breast cancer with high UPR was significantly associated with worse survival, but neither HER-positive nor TNBC. High UPR ER-positive/HER2-negative breast cancer was infiltrated with high level of Th1 and Th2 cells, M1 macrophage, and plasma cells. On the other hand, they were significantly infiltrated with high level of several types of stromal cells in tumor microenvironment (all P<0.001). Finally, high UPR metastatic breast cancer was also associated with worse patient survival (P=0.041). UPR signaling is associated with cancer aggressiveness, and worse survival, especially ER-positive/HER2-negative breast cancer subtype.