LASP-1 was identified as a protein following mass spectrometric analysis of phosphoproteins consequent to signaling by ErbB2 in SKOV-3 cells. It has been previously identified as an oncogene and is located on chromosomal arm 17q 0.76 Mb centromeric to ErbB2. It is expressed in serous ovarian cancer cell lines as a 40 kDa protein. In SKOV-3 cells, it was phosphorylated and was inhibited by Lapatinib and CP7274714. LASP-1 co-immunoprecipitated with ErbB2 in SKOV-3 cells, suggesting a direct interaction. This interaction and phosphorylation were independent of the kinase activity of ErbB2. Moreover, the binding of LASP-1 to ErbB2 was independent of the tyrosine phosphorylation of LASP-1. LASP-1 was neither expressed on the surface epithelium of the normal ovary nor in the fallopian tube. It was expressed in 28% of ovarian tumours (n =101) that did not significantly correlate with other clinical factors. In tumours from patients with invasive ductal carcinoma of the breast who had ErbB2 amplification (3+), LASP-1 was expressed in 3/20 (P < 0.001). Analysis of the expression of an independent dataset of ovarian and breast tumours from TCGA showed the significant co-occurrence of ErbB2 and LASP-1 (P < 0.01). These results suggest that LASP-1 and ErbB2 interaction could be important in the pathogenesis of ovarian cancer.

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.

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.

Ovarian cancer is the most common cause of mortality among gynecological cancers, despite advances in treatment. Recurrence is common and is due to development of drug resistance. One of the reasons for drug resistance is the persistence of cancer stem cells. To understand the role of CSCs, it is essential to capture and propagate cells continuously in culture. Reprogramming cancer cells to induced pluripotent stem cells (iPSCs) is an approach to achieve this. An ovarian cancer cell line, PEO4 (high grade serous adenocarcinoma), was initially reprogrammed into iPSCs using the classical four factors OCT4, SOX2, KLF4 and MYC (OSKM) using STEMCCA by lentivirus transduction. Embryonic stem cell (ESC)-like bodies appeared between 8 to 15 days post-transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. Two individual clones were further characterized. The reprogrammed PEO4-OSKM-iPSCs expressed alkaline phosphatase and pluripotency markers, NANOG, OCT4, SSEA4, TRA-1-60 and TRA-1-81 by immunofluorescence. Further, reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed expression of the pluripotency markers NANOG, SOX2, OCT4, TERT, NESTIN, DMNT, DPPA4 in PEO4-OSKM-iPSC which were absent in the parental PEO4 cells. PEO4-OSKM-iPSC cells could be differentiated in vitro with appropriate growth factors into ectodermal, mesodermal and endodermal lineages. MYC was replaced with GLIS1 in the lentiviral cassette and PEO4 cells were able to be transformed to iPSCs. The transfection efficiency was two fold better with OCT4-SOX2-KLF4-GLIS1 with larger colonies. Individual iPSC colonies expressed all pluripotency markers and were able to differentiate into all 3 lineages. Characterization of iPSC cells for expression of cell surface markers specific for serous adenocarcinoma, showed that CD133, EPHA1, CD44 and LGR5 were expressed. Cell viability assays demonstrated that IC50 of cisplatin in parental PEO4 cells (15uM) was less as compared to iPSC cells (32uM) (p<0.03) and similarly, IC50 of paclitaxel was less in parental PEO4 (17 uM) as compared to iPSC cells (27 uM) (p<0.02). These results demonstrate for the first time that an ovarian cancer cell line derived from a patient with high grade serous adenocarcinoma can be reprogrammed. Further, GLIS1 can successfully replace MYC as a transcription factor to generate induced pluripotent stem cells. Citation Format: S Bindhya, C Sidhanth, S Krishnapriya, R P. Nagare, M Garg, T S. Ganesan. GLIS1 can replace MYC to generate induced pluripotent stem cells from ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3693.

The origin of blood and lymphatic vessels in high-grade serous adenocarcinoma of ovary (HGSOC) is uncertain. We evaluated the potential of cancer stem cells (CSCs) in HGSOC to contribute to their formation. Using spheroids as an in vitro model for CSCs, we have evaluated their role in primary malignant cells (PMCs) in ascites from previously untreated patients with HGSOC and cell lines. Spheroids from PMCs grown under specific conditions showed significantly higher expression of endothelial, pericyte and lymphatic endothelial markers. These endothelial and lymphatic cells formed tube-like structures, showed uptake of Dil-ac-LDL and expressed endothelial nitric oxide synthase confirming their endothelial phenotype. Electron microscopy demonstrated classical Weibel–Palade bodies in differentiated cells. Genetically, CSCs and the differentiated cells had a similar identity. Lineage tracking using green fluorescent protein transfected cancer cells in nude mice confirmed that spheroids grown in stem cell conditions can give rise to all three cells. Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor inhibited the differentiation of spheroids to endothelial cells in vitro. These results suggest that CSCs contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary, which can be inhibited.

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.

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.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies having no effective treatment. Exportin-1 (XPO1) is the key mediator of nuclear export of many tumor suppressor proteins and is overexpressed in human cancers. In this study, we examined the therapeutic potential of selinexor (XPO1 inhibitor) against human ATC cells both in vitro and in vivo . Here, we showed that XPO1 is robustly expressed in primary ATC samples and human ATC cell lines. Silencing of XPO1 by either shRNA or selinexor significantly reduced cellular growth and induced cell cycle arrest, apoptosis of ATC cells by altering the protein expression of cancer-related genes. Moreover, selinexor significantly inhibited tumor growth of ATC xenografts. Microarray analysis showed enrichment of DNA replication, cell cycle, cell cycle checkpoint and TNF pathways in selinexor treated ATC cells. Importantly, selinexor decreased AXL and GAS6 levels in CAL62 and HTH83 cells and suppressed the phosphorylation of downstream targets of AXL signaling such as AKT and P70S6K. Finally, a combination of selinexor with doxorubicin demonstrated a synergistic decrease in the cellular proliferation of several ATC cells. These results provide a rationale for investigating the efficacy of combining selinexor and doxorubicin therapy to improve the outcome of ATC patients.

Abstract The receptor tyrosine kinase c-ErbB2 is amplified in breast and ovarian cancer. The linear pathways through which signals by c-ErbB2 are transduced is 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. We have used two tyrosine kinase inhibitors, Lapatinib and CP724714, that inhibit phosphorylation of c-ErbB2 to identify phosphoproteins. SKOV-3, an ovarian cancer cell line that endogenously overexpresses c-ErbB2 was grown in culture without serum for 72 hrs. Cells were then stimulated in the presence or absence of inhibitor with EGF (100ng/ml) as a ligand for 60 mins. Subsequently, cells were lysed and evaluated by western blotting with anti-phosphotyrosine antibody (4G10). Following stimulation of cells with EGF, maximal phosphorylation of c-ErbB2 was observed at 60 minutes. Lapatinib (10μM) and CP724714 (15μM) completely inhibited phosphorylation of c-ErbB2, which was confirmed by immunoprecipitation. This was further confirmed by the inhibition of downstream effectors (Erk1/2, Akt). Lapatinib (10 μM) also completely inhibited phosphorylation of EGFR while CP724714 (15μM) only inhibited partially. Cellular lysates were prepared from quiescent cells (grown without serum), after stimulation with EGF in the presence or absence of inhibitors. Purified phosphoproteins from all three samples following digestion with trypsin were subjected to mass spectrometry (Nano LC ESI MS/MS). We identified totally 62 phosphoproteins. Twenty seven phosphoproteins were observed in all the 3 samples while 17 phosphoproteins were identified both in the EGF stimulated and lapatinib treated samples. Eighteen unique phosphoproteins were observed only in the EGF stimulated sample suggesting that they are specific to signaling by c-ErbB2. The novel phosphoproteins included the proteins that partcipate in carbohydrate metabolism,cytoskeleton, cell migration and proliferation. We have evaluated two phosphoproteins, LASP-1 and Aldose reductase that has not been previously described following phosphorylation of c-ErbB2. LASP-1 is an oncogene and is located as the same arm 17q21 as c-ErbB2. It was not expressed in the normal ovary or fallopian tube. However, it was over-expressed in 17% of tumours (n=85) from patients with ovarian cancer. c-ErbB2 was not expressed in tumours that expressed LASP1. Aldose reductase is a cytosolic NADPH dependent oxidoreductase that catalyzes the reduction of glucose to sorbitol, the first step in polyol pathway of glucose metabolism. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in lysates from EGF stimulated as compared to the starved cells. Identification of phosphoproteins by using mass spectrometry is promising in identifying novel substrates and pathways following phosphorylation of c-ErbB2. Citation Format: C Sidhanth, Manoj Garg, P Manasa, S Krishna Priya, S Bindhya, S Sneha, R.P. Nagare, S Shirley, M Kanchan, Trivadi S. Ganesan. Identification by mass spectrometry of unique phosphoproteins subsequent to signaling through c-ErbB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 212. doi:10.1158/1538-7445.AM2017-212

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.

Cancer is one of the leading cause of morbidity and mortality worldwide. Regulated nucleo-cytoplasmic shuttling is very crucial for maintaining cellular homeostasis. Emerging evidence suggests that deregulation of the nucleo-cytoplasmic transport results in abnormal cell growth, cell cycle, apoptosis, tumor progression and drug resistance. Exportin-1 (also called as chromosome region maintenance 1) belongs to karyopherin-β superfamily and is the main mediator of nuclear export in several cell types. The XPO1 / CRM1 protein is overexpressed in liposarcoma, Ewing sarcoma, ovarian carcinoma, pancreatic cancer, hepatocellular carcinoma, lung carcinoma, osteosarcoma, gastric carcinoma, melanoma, glioma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid/lymphoid leukemia as well as multiple myeloma. Hot spot mutations were observed in many cancers. Higher levels of XPO1 / CRM1 are associated with poor prognosis, resistance to chemotherapy and recurrence in a large number of human malignancies. There are growing evidence that provided the foundation that inhibition of nuclear export by inhibiting nuclear export receptor ( XPO1 ) might be a potential targeted therapeutic approach for the treatment of human cancers in the clinic. In the present review, we will discuss the role of XPO1 in cancers and potential of selective inhibitors of nuclear export ( XPO1 inhibitors) to restore the normal function of tumor suppressor and growth regulatory proteins by blocking their export. Selinexor (KPT-330) is an orally available, highly potent and is being tested in human phase-I/II clinical trials in both haematological and solid malignancies.

The traditional view of tumour vascularization is that tumours acquire blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSCs) in glioblastoma. In high grade serous ovarian cancer (HGSOC), the origin of endothelial cells is not known. Our objective was to determine if components of a tumour blood vessel and lymphatic vessel are derived from CSCs in ovarian cancer. Using spheroids as an in vitro model, we have evaluated the role of CSCs in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary cultured under specific conditions. The expression of endothelial, pericyte and lymphatic endothelial markers was evaluated by flow cytometry. In addition, functional assays were performed to assess the endothelial phenotype. Further, the ability of CSCs to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab which antagonize VEGF. PMCs grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (n=32, p = 0.002) and CLEC14A (n=10, p = 0.012) and co-expression of CD105/CLEC14A (n=10, p= 0.012) than that of spheroids. Primary malignant cells when grown in pericyte and lymphatic endothelial specific conditions, showed significantly higher expression of desmin (n=10, p=0.03), Smooth muscle actin (SMA) (n=10, p=0.017) and VEGFR3 (n=10, p=0.028) than that of the spheroids. When the PMCs were grown as spheroids in endothelial conditions in the presence or absence of Bevacizumab (1 μg/μl), there was a reduction in the co-expression of CD105 and CLEC14A (P=0.04). The cells grown in endothelial conditions showed formation of tubes, uptake of Dil-ac-LDL and expressed eNOS, confirming their endothelial phenotype. GFP transduced spheroids from PMCs formed tumours in mice and the blood vessels in the tumour co-expressed CD31, SMA, VEGFR3 and GFP, suggesting that these cells are derived from CSCs. These results prove that a proportion of endothelial cells, pericytes and lymphatic endothelial cells are derived from CSCs in serous ovarian carcinoma and the VEGF pathway has a key role. This property of CSCs to contribute to tumour angiogenesis can be inhibited.Citation Format: S Krishnapriya, C Sidhanth, P Manasa, S Sneha, S Bindhya, R P. Nagare, T S. Ganesan. Cancer stem cells and tumor angiogenesis in serous adenocarcinoma of ovary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 182.

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.

In this study, the micronucleus (MN) frequency was assessed as a measure of genotoxicity in exfoliated cells of buccal mucosa extracted from 110 petrol pump workers and 100 controls. For each individual, 3,000 exfoliated buccal cells were analyzed. The individuals used in the study were grouped based on their smoking, drinking alcoholic beverages, and tobacco chewing habits. There was a significantly higher frequency of micronucleated cells in the exposed workers to petrol than in the unexposed control population. Smoking and drinking (alcohol) habits, age and length of occupation represent significant factors in terms of increasing the MN frequency measured in the exposed population. This study demonstrates that, using MN assay, it is possible to assess the cytogenetic damage in exposed individuals and that the significant increase in the induction of the MN in the exposed population suggests that the studied individuals may be at a higher risk of developing cancer and therefore monitored for any long term adverse effects of the exposure.

The focal aim of this study was to assess the frequency of chromosomal aberrations (CA) including chromatid type aberrations (CTA) and chromosomal type aberrations (CSA), micronucleus (MN) and XRCC1 399 Arg/Gln polymorphism in the peripheral blood lymphocytes of 27 petrol pump workers and same number of controls to explore the possible cytogenetic risk on occupational exposure to petrol vapors. The exposed subjects and controls were classified into two groups based on their age (group I < 40 years; group II > 40 years) apart from the classification of the exposed subjects based on their exposure duration (> 8 and < 8 years). CTA and MN frequency were significantly higher in petrol pump workers (p < 0.05) with longer work duration. CTA was found to increase with age in the exposed subjects as well as controls, with exposed subjects showing a statistically higher degree. This effect was not observed in MN. A significantly higher frequency of MN was observed in the smoking petrol pump workers than in control smokers (p < 0.05). No association was found between smoking and CA in both subjects. The study on XRCC1 399 Arg/gln polymorphism in petrol pump workers demonstrated very less difference in allele frequency compared to controls. In conclusion, these datas indicate that petrol pump workers under risk group should be monitored for any long-term adverse effects of the exposure.