Phosphoproteome of signaling by ErbB2 in ovarian cancer cells.

The gene for receptor tyrosine kinase ErbB2 is amplified in breast and ovarian tumours. The linear pathway by which signals are transduced through ErbB2 are well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins specific for ErbB2 using the inhibitors Lapatinib and CP724714 in ovarian cancer cells. The ErbB2 specific proteins identified in SKOV-3 cells were Myristoylated alanine-rich C-kinase substrate, Protein capicua homolog, Protein peptidyl isomerase G, Protein PRRC2C, Chromobox homolog1 and PRP4 homolog. We have evaluated three phosphoproteins PKM2, Aldose reductase and MARCKS in SKOV-3 cells. We observed that PKM2 was phosphorylated by EGF but was not inhibited by Lapatinib and CP724714. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in EGF stimulated cells which was inhibited by Lapatinib and CP724714 but not by Geftinib (EGFR inhibitor). MARCKS was phosphorylated on stimulation of SKOV-3 cells with EGF that was inhibited by Lapatinib and CP724714 which was dependent on the kinase activity of ErbB2. These results have identified phosphoproteins that are specific to ErbB2 which have not been previously reported and sets the basis for future experiments.

Development and in vitro characterisation of an induced pluripotent stem cell model of ovarian cancer.

Ovarian cancer recurs despite advances in treatment and is due to drug resistance. The persistence of cancer stem cells (CSCs) is one of the causes. It has been challenging to maintain CSCs long term in culture from primary malignant cells. Reprogramming cancer cells into induced pluripotent stem cells (iPSCs) could be an approach to achieve this. An ovarian cancer cell line, PEO4, was initially reprogrammed into iPSCs using the classical four factors OCT4, SOX2, KLF4 and MYC (OSKM) using lentivirus transduction. The PEO4-OSKM-cells had all the hallmarks of iPSCs. As MYC is oncogenic, we have replaced it with GLIS1 and show that PEO4 cells could be transformed into iPSCs. The transfection efficiency was two-fold better with OCT4-SOX2-KLF4-GLIS1 (OSKG) with larger colonies. Further, normal fallopian tube epithelial cells were also transformed using OSKG into iPSCs. iPSCs expressed CSCs markers such as CD133, EPHA1, ALDH1A1 and LGR5 prominently and were more resistant to cisplatin and taxol as compared to parental PEO4 cells. PEO4-OSKM-iPSCs cells formed more colonies in a clonogenic assay as compared to PEO4-OSKG-iPSCs and parental cells. These results provide a first insight that both an ovarian cancer cell line and fallopian tube epithelial cells can be reprogrammed and GLIS1 can successfully replace MYC as a transcription factor. This in vitro model is useful for future experiments to understand the characteristics of CSCs in the pathogenesis of ovarian cancer.

Induced pluripotent stem cells: A new strategy to model human cancer.

Induced pluripotent stem cells are derived from adult somatic cells by ectopic expression of stem cell factors OCT4, SOX2, MYC and KLF4. These cells have characteristic features similar to embryonic stem cells. Although there exists in vitro and in vivo models of cancer, recapitulating the earliest events in the pathogenesis remain challenging. More recently, induced pluripotent stem cells have been generated to model human disease and cancer. There are advantages in the cancer models derived from these cells as compared to existing conventional approaches. Induced pluripotent stem cells have been generated from cancer cell lines, primary tumours and from those with an inherited predisposition to develop cancer. In addition, these cells provide a valuable tool in understanding the pathogenesis of familial cancer in its earliest stages, and to identify additional genetic alterations that are required to develop cancer. Furthermore, these cells can serve as a resource in drug screening and developing new therapies.

A systematic understanding of signaling by ErbB2 in cancer using phosphoproteomics.

ErbB2 is an important receptor tyrosine kinase and a member of the ErbB family. Although it does not have a specific ligand, it transmits signals downstream by heterodimerization with other receptors in the family. It plays a major role in a variety of cellular responses like proliferation, differentiation, and adhesion. ErbB2 is amplified at the DNA level in breast cancer (20%-30%) and gastric cancer (10%-20%), and trastuzumab is effective as a therapeutic antibody. This review is a critical analysis of the currently published data on the signaling pathways of ErbB2 and the interacting proteins. It also focuses on the techniques that are currently available to evaluate the entire phosphoproteome following activation of ErbB2. Identification of new and relevant phosphoproteins can not only serve as new therapeutic targets but also as a surrogate marker in patients to assess the activity of compounds that inhibit ErbB2. Overall, such analysis will improve understanding of signaling by ErbB2.

Evaluation of a polymorphism in MYBPC3 in patients with anthracycline induced cardiotoxicity.

Cardiotoxicity is the most serious side effect of anthracyclines (doxorubicin, daunorubicin or epirubicin). The incidence of anthracycline induced late cardiac toxicity (AIC) that is overt clinically is 3-5% in the Indian population. Polymorphism in intron 32 (deletion of 25bp) of MYBPC3 has been shown to be present exclusively in Asians and more so in South India (3-8%). The frequency of the polymorphism is significantly higher (13%) in patients with cardiomyopathy in India. Fifteen patients were identified to have cardiac dysfunction following treatment for malignant lymphoma with doxorubicin containing regimens. Peripheral blood DNA from control, amplified by polymerase chain reaction yielded a 467bp fragment while in the presence of the 25bp deletion only a 442bp fragment was detected. To confirm the presence or absence of the polymorphism, amplified DNA was restricted using Bgl1 in all samples. Bgl1 restricted amplified DNA only if the 25bp deletion was absent. A 467 base pair band was observed in all the 15 samples, which suggested the absence of polymorphism in MYBPC3. In a sample of DNA from a patient with a deletion in exon 33 (confirmed by sequencing) a 442bp fragment was detected. Amplified DNA from this patient was not restricted with Bgl1. Wild type MYBPC3 when amplified gave a distinct restriction banding pattern consisting of two bands of 401bp and 66bp. Amplified DNA from all peripheral blood samples restricted with Bgl1 suggesting the absence of the polymorphism. In this preliminary report, MYBPC3 does not seem to play a role in anthracycline induced cardiotoxicity.

Tumour angiogenesis-Origin of blood vessels.

The conventional view of tumour vascularization is that tumours acquire their blood supply from neighbouring normal stroma. Additional methods of tumour vascularization such as intussusceptive angiogenesis, vasculogenic mimicry, vessel co-option and vasculogenesis have been demonstrated to occur. However, the origin of the endothelial cells and pericytes in the tumour vasculature is not fully understood. Their origin from malignant cells has been shown indirectly in lymphoma and neuroblastoma by immuno-FISH experiments. It is now evident that tumours arise from a small population of cells called cancer stem cells (CSCs) or tumour initiating cells. Recent data suggest that a proportion of tumour endothelial cells arise from cancer stem cells in glioblastoma. This was demonstrated both in vitro and in vivo. The analysis of chromosomal abnormalities in endothelial cells showed identical genetic changes to those identified in tumour cells. However, another report contradicted these results from the earlier studies in glioblastoma and had shown that CSCs give rise to pericytes and not endothelial cells. The main thrust of this review is the critical analysis of the conflicting data from different studies and the remaining questions in this field of research. The mechanism by which this phenomenon occurs is also discussed in detail. The transdifferentiation of CSCs to endothelial cells/pericytes has many implications in the progression and metastasis of the tumours and hence it would be a novel target for antiangiogenic therapy.