Efficacy of Lapatinib in Therapy-Resistant HER2-Positive Circulating Tumor Cells in Metastatic Breast Cancer

Background

To evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in therapy-resistant HER2-positive CTCs in metastatic breast cancer (MBC).

Patients and Methods

Patients with MBC and HER2-positive CTCs despite disease stabilization or response to prior therapy, received lapatinib 1500 mg daily in monthly cycles, till disease progression or CTC increase. CTC monitoring was performed by immunofluorescent microscopy using cytospins of peripheral blood mononuclear cells (PBMCs) double stained for HER2 or EGFR and cytokeratin.

Results

A total of 120 cycles were administered in 22 patients; median age was 62.5 years, 15 (68.2%) patients were post-menopausal and 20 (90.1%) had HER2-negative primary tumors. At the end of the second course, HER2-positive CTC counts decreased in 76.2% of patients; the median number of HER2-positive CTCs/patient also declined significantly (p = 0.013), however the decrease was significant only among patients presenting disease stabilization (p = 0.018) but not among those with disease progression during lapatinib treatment. No objective responses were observed. All CTC-positive patients harbored EGFR-positive CTCs on progression compared to 62.5% at baseline (p = 0.054). The ratio of EGFR-positive CTCs/total CTCs detected in all patients increased from 17.1% at baseline to 37.6% on progression, whereas the mean percentage of HER2-negative CTCs/patient increased from 2.4% to 30.6% (p = 0.03).

Conclusions

The above results indicate that lapatinib is effective in decreasing HER2-positive CTCs in patients with MBC irrespectively of the HER2 status of the primary tumor and imply the feasibility of monitoring the molecular changes on CTCs during treatment with targeted agents.

Trial Registration

Clinical trial.gov NCT00694252     

Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells.

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist alpha(s1) casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and alpha (s1) casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.     

Distinct signaling pathways regulate differential opioid effects on actin cytoskeleton in malignant MCF7 and nonmalignant MCF12A human breast epithelial cells

The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.     

Common Players in Mitochondria Biogenesis and Neuronal Protection Against Stress-Induced Apoptosis

Mitochondria biogenesis is a fundamental process for the organization and normal function of all cells. Since the majority of mitochondrial proteins are synthesized in the cytosol, protein import is the major mechanism for mitochondria biogenesis. We describe the different pathways that ensure correct targeting and intra mitochondrial sorting of mitochondrial proteins. The import process of several proteins of the mitochondrial intermembrane space relies on the Mitochondrial Import and Assembly 40 and Essential for respiration and vegetative growth 1 (Erv1) proteins that together constitute the oxidative folding machinery of the mitochondrial intermembrane space. Recent work has implicated the FAD-oxidase protein Erv1 (ad its human homolog Augmenter of Liver Regeneration) as an anti-apoptotic factor in mammalian cells (including neuronal cells) that undergo Reactive Oxygen Species-triggered apoptosis. The different roles of this protein as a key factor in mitochondria biogenesis, iron-sulfur cluster biogenesis and in neuronal protection against apoptosis are discussed.     

Targeting and maturation of Erv1/ALR in the mitochondrial intermembrane space

The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the intermembrane space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40. The action of Mia40 is selective for the formation of both intra and intersubunit structural disulfide bonds of Erv1/ALR, but the complete maturation process requires additional binding of FAD. Both of these events must follow a specific sequential order to allow Erv1/ALR to reach the fully functional state, illustrating a new paradigm for protein maturation in the IMS.     

Phosphorylation of FAK, PI-3K, and impaired actin organization in CK-positive micrometastatic breast cancer cells

Several markers have been used to detect circulating tumor cells (CTC) in the peripheral blood of patients with breast cancer. However, analysis of activated signaling kinases in CTC implicated in cellular transformation, migration, and survival has not been addressed so far. In the present study, we focused on the phenotypic profile of micrometastatic cells in peripheral blood mononuclear cells (PBMC) preparations from 45 breast cancer patients. PBMC cytospins from 28 cytokeratin (CK)-positive and 17 CK-negative samples were assessed for the expression of phosphorylated FAK (p-FAK), phosphorylated PI-3 kinase (p-PI-3K), and HER2 using confocal laser scanning microscopy. The expression of p-FAK was documented in all 28 CK-positive samples, while all 17 CK-negative samples were tested negative for p-FAK. Immunomagnetic separation using EpCAM antibody fully confirmed these findings, implying a sound correlation for the co-expression of the two molecules. Interestingly, 15 of 28 CK- and p-FAK-positive samples also expressed the HER2 oncoprotein. p-PI-3K was documented in 15 of 17 CK- and p-FAK-positive samples. Immunoblot analysis of micrometastatic cells in co-culture with PBMC confirmed the specific expression of both p-FAK and p-PI-3K. Finally, impaired actin organization was apparent in CK- and p-FAK/p-PI-3K-positive samples, comparable to that observed in MCF-7 human breast cancer cells. Our findings provide strong evidence that micrometastatic cells express activated signaling kinases, which may regulate migration mechanisms, supporting the presumption of their malignant and metastatic nature.     

Context matters: On the road to responsible biosafety technologies in synthetic biology

Biosafety is a major challenge for developing for synthetic organisms. An early focus on application and their context could assist with the design of appropriate genetic safeguards. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business

One of the goals of synthetic biology is the development of robust chassis cells for their application in medicine, agriculture, and the food, chemical and environmental industries. These cells can be streamlined by removing undesirable features and can be augmented with desirable functionalities to design an optimized organism. In a direct analogy with a car chassis, they provide the frame for different modules or “plug‐in” regulatory networks, metabolic pathways, or safety elements. In an effort to ensure a safe microbial chassis upfront, safety measures are implemented as genetic safeguards to limit risks such as unwanted cellular proliferation or horizontal gene transfer. Examples of this technology include complex genetic circuits, sophisticated metabolic dependencies (auxotrophies), and altered genomes (Schmidt & de Lorenzo, 2016; Asin‐Garcia et al, 2020). Much like seat belts or airbags in cars, these built‐in measures increase the safety of the chassis and of any organisms derived from it. Indeed, when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.Every car today has seat belts installed by default. Yet, seat belts were not always a standard component; in fact, they were not even designed for cars to begin with. The original 2‐point belts were first used in aviation and only slowly introduced for motorized vehicles. Only after some redesign, the now‐common 3‐point car seat belts would become the life‐saving equipment that they are today. A proper understanding of the context of their application was therefore one of the crucial factors for their success and wide adoption. Context matters: It provides meaning for and defines what a technological application is best suited for. What was true for seat belts may be also true for biosafety technologies such as genetic safeguards.

… when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.

Society has a much higher awareness of technology’s risks compared to the early days of cars. Society today requires that technological risks are anticipated and assessed before an innovation or its applications are widely deployed. In addition, society increasingly demands that research and innovation take into account societal needs and values. This has led to, among others, the Responsible Research and Innovation (RRI; von Schomberg, 2013) concept that has become prominent in European science policy. In a nutshell, RRI requires that innovative products and processes align with societal needs, expectations, and values in consultation with stakeholders. RRI and similar frameworks suggest that synthetic biology must anticipate and respond not only to risks, but also to societal views that frame its evaluation and risk assessment.