Immunoliposomal delivery of doxorubicin can overcome multidrug resistance mechanisms in EGFR-overexpressing tumor cells.

Immunoliposomes (ILs) can be constructed to target the epidermal growth factor receptor (EGFR) to provide efficient intracellular drug delivery in tumor cells. We hypothesized that this approach might be able to overcome drug resistance mechanisms, which remain an important obstacle to better outcomes in cancer therapy. ILs were evaluated in vitro and in vivo against EGFR-overexpressing pairs of human cancer cells (HT-29 and MDA-MB-231) that either lack or feature the multidrug resistance (mdr) phenotype. In multidrug-resistant cell lines, ILs loaded with doxorubicin (DOX) produced 19-216-fold greater cytotoxicity than free DOX, whereas in nonresistant cells, immunoliposomal cytotoxicity of DOX was comparable with that of the free drug. In intracellular distribution studies, free DOX was efficiently pumped out of the multidrug-resistant tumor cells, whereas immunoliposomal DOX leads to 3.5-8 times higher accumulation of DOX in the cytoplasm and 3.5-4.9 times in the nuclei compared with the free drug. Finally, in vivo studies in the MDA-MB-231 Vb100 xenograft model confirmed the ability of anti-EGFR ILs-DOX to efficiently target multidrug-resistant cells and showed impressive antitumor effects, clearly superior to all other treatments. In conclusion, ILs provide efficient and targeted drug delivery to EGFR-overexpressing tumor cells and are capable of completely reversing the multidrug-resistant phenotype of human cancer cells.

Increased Level of Phosphorylated ShcA Measured by Chemiluminescence-Linked Immunoassay Is a Predictor of Good Prognosis in Primary Breast Cancer Expressing Low Levels of Estrogen Receptor.

The SH2 domain-containing adaptor protein ShcA is a proto-oncogene involved in growth factor receptor signaling. The role of phosphorylated ShcA is to link receptor tyrosine kinases with the SH2-containing adaptor protein Grb2, thus facilitating signal transduction from receptor tyrosine kinases to Ras, leading to MAPK activation. The present study was designed to investigate the prognostic significance of phosphorylated ShcA in primary breast cancer and its association in the interactions between the ER and ErbB2 pathways. Using a two-site chemiluminescence-linked immunosorbent assay, we detected the quantitative expression levels of total tyrosine- and threonine-phosphorylated ShcA in cytosol fractions obtained from fresh frozen tissue samples of 153 selected primary breast cancer patients. ShcA phosphorylation was not associated with nodal status, estrogen receptor (ER) status or grading. High levels of both tyrosine (pYShcA) and serine (pSShcA) phosphorylated ShcA correlated with good prognosis (p < 0.01), with respect to both disease-free (DFS) and overall survival (OS). In addition, pShcA levels were found to correlate with threonine-phosphorylated ErbB2 and inversely with phosphorylated Akt (pAkt), as well as ErbB2 and ER expression levels. Our findings demonstrate that ShcA activation in primary breast cancer patients correlates with low levels of ER, and is associated with good prognosis.

EGFR-targeted immunoliposomes derived from the monoclonal antibody EMD72000 mediate specific and efficient drug delivery to a variety of colorectal cancer cells.

We hypothesized that immunoliposomes (ILs) constructed using Fab' from the humanized anti-EGFR monoclonal antibody, EMD72000, can provide efficient intracellular drug delivery in EGFR-overexpressing colorectal tumor cells.ILs were constructed modularly with various MAb fragments, including Fab' from EMD72000 (matuzumab) or C225 (cetuximab, Erbitux) covalently linked to stabilized liposomes containing chemotherapeutic drugs or probes. Immunoliposome preparation was optimized, including Fab' reduction and linkage, and evaluated for specific binding and cytotoxicity in epidermal growth factor receptor (EGFR)--overexpressing colorectal cancer cell lines in vitro. Flow cytometry showed that EGFR-targeted ILs, but not non-targeted liposomes or irrelevant ILs, were efficiently bound and internalized by a variety of EGFR-overexpressing colorectal cancer cells. Linkage of the Fab' to a longer PEG chain (Mal-PEG3400-DSPE) resulted in an increased uptake of immunoliposomal constructs when compared to previously used materials (Mal-PEG2000-DSPE). ILs derived from EMD72000-Fab' were used to deliver doxorubicin to EGFR-overexpressing target cells in vitro. Immunoliposomal doxorubicin was significantly more cytotoxic than the corresponding non-targeted liposomal drug in target cells, such as HCT116, while equivalent in cells lacking EGFR-overexpression, such as Colo205. We conclude that EGFR-targeted ILs derived from the humanized MAb EMD72000 provide efficient and targeted delivery of anticancer drugs in colorectal cancer cells overexpressing EGFR.

Heregulins implicated in cellular functions other than receptor activation.

Heregulins (HRG) are known as soluble secreted growth factors that, on binding and activating ErbB3 and ErbB4 cell surface receptors, are involved in cell proliferation, metastasis, survival, and differentiation in normal and malignant tissues. Previous studies have shown that some HRG1 splice variants are translocated to the nucleus. By investigating the subcellular localization of HRGalpha(1-241), nuclear translocation and accumulation in nuclear dot-like structures was shown in breast cancer cells. This subcellular distribution pattern depends on the presence of at least one of two nuclear localization sequences and on two domains on the HRG construct that were found to be necessary for nuclear dot formation. Focusing on the nuclear function of HRG, a mammary gland cDNA library was screened with the mature form of HRGalpha in a yeast two-hybrid system, and coimmunoprecipitation of endogenous HRG was done. The data reveal positive interactions of HRGalpha(1-241) with nuclear factors implicated in different biological functions, including transcriptional control as exemplified by interaction with the transcriptional repressor histone deacetylase 2. In addition, HRGalpha(1-241) showed transcriptional repression activity in a reporter gene assay. Furthermore, a potential of HRG proteins to form homodimers was reported and the HRG sequence responsible for dimerization was identified. These observations strongly support the notion that HRG1 splice variants have multifunctional properties, including previously unknown regulatory functions within the nucleus that are different from the activation of ErbB receptor signaling.

Phosphorylation of tyrosine 1248-ERBB2 measured by chemiluminescence-linked immunoassay is an independent predictor of poor prognosis in primary breast cancer patients.

ERBB2 (HER2/Neu) gene amplification and overexpression is associated with increased risk of metastases and shorter survival in breast cancer. Tyrosine 1248 is a major phosphorylation site of ERBB2 and reflects the activation status of the receptor. The aim of this study was to investigate the relationships between quantitative levels of pY1248-ERBB2 (p-ERBB2) and the expression of epidermal growth factor receptor (EGFR)-family members, and whether p-ERBB2 could provide additional prognostic value compared with established prognostic markers. For this purpose we developed a highly sensitive chemiluminescence-linked immunoassay (CLISA) and detected p-ERBB2 levels in 70 primary breast cancer biopsies. Phosphorylated ERBB2 correlated with EGFR and ERBB2, and inversely with oestrogen receptor (ER), progesterone receptor (PgR) and ERBB4 expression levels. Additionally, p-ERBB2 was associated with poor clinical outcome in univariate and multivariate Cox regression analysis. Further studies are needed to evaluate the predictive value of p-ERBB2.

Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2.

INTRODUCTION: Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers. METHODS: Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients. RESULTS: Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR. CONCLUSION: Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours.