Vaijayanti Gupta

A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

A Global In Vivo Drosophila RNAi Screen Identifies NOT3 as a Conserved Regulator of Heart Function

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animals survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Role of the Drug Transporter ABCC3 in Breast Cancer Chemoresistance

Increased expression of ABC-family of transporters is associated with chemotherapy failure. Although the drug transporters ABCG2, ABCB1 and ABCC1 have been majorly implicated in cancer drug resistance, recent studies have associated ABCC3 with multi drug resistance and poor clinical response. In this study, we have examined the expression of ABCC3 in breast cancers and studied its role in drug resistance and stemness of breast cancer cells in comparison with the more studied ABCC1. We observed that similar to ABCC1, the transcripts levels of ABCC3 was significantly high in breast cancers compared to adjacent normal tissue. Importantly, expression of both transporters was further increased in chemotherapy treated patient samples. Consistent with this, we observed that treatment of breast cancer cell lines with anti-cancer agents increased their mRNA levels of both ABCC1 and ABCC3. Further, similar to knockdown of ABCC1, knockdown of ABCC3 also significantly increased the retention of chemotherapeutic drugs in breast cancer cells and rendered them more chemo-sensitive. Interestingly, ABCC1 and ABCC3 knockdown cells also showed reduction in the expression of stemness genes, while ABCC3 knockdown additionally led to a reduction in the CD44high/CD24low breast cancer stem-like subpopulation. Consistent with this, their ability to form primary tumours was compromised. Importantly, down-modulation of ABCC3 rendered these cells increasingly susceptible to doxorubicin in xenograft mice models in vivo. Thus, our study highlights the importance of ABCC3 transporters in drug resistance to chemotherapy in the context of breast cancer. Further, these results suggest that combinatorial inhibition of these transporters together with standard chemotherapy can reduce therapy-induced resistance in breast cancer.

StrandAdvantage test for early-line and advanced-stage treatment decisions in solid tumors

Comprehensive genetic profiling of tumors using next‐generation sequencing (NGS) is gaining acceptance for guiding treatment decisions in cancer care. We designed a cancer profiling test combining both deep sequencing and immunohistochemistry (IHC) of relevant cancer targets to aid therapy choices in both standard‐of‐care (SOC) and advanced‐stage treatments for solid tumors. The SOC report is provided in a short turnaround time for four tumors, namely lung, breast, colon, and melanoma, followed by an investigational report. For other tumor types, an investigational report is provided. The NGS assay reports single‐nucleotide variants (SNVs), copy number variations (CNVs), and translocations in 152 cancer‐related genes. The tissue‐specific IHC tests include routine and less common markers associated with drugs used in SOC settings. We describe the standardization, validation, and clinical utility of the StrandAdvantage test (SA test) using more than 250 solid tumor formalin‐fixed paraffin‐embedded (FFPE) samples and control cell line samples. The NGS test showed high reproducibility and accuracy of >99%. The test provided relevant clinical information for SOC treatment as well as more information related to investigational options and clinical trials for >95% of advanced‐stage patients. In conclusion, the SA test comprising a robust and accurate NGS assay combined with clinically relevant IHC tests can detect somatic changes of clinical significance for strategic cancer management in all the stages.

Expression of tripartite motif-containing protein 28 in primary breast carcinoma predicts metastasis and is involved in the stemness, chemoresistance, and tumor growth:

The prediction of who develops metastasis has been the most difficult aspect in the management of breast cancer patients. The lymph node metastasis has been the most useful predictor of prognosis and patient management. However, a good proportion of patients with lymph node positivity remain disease free for 5 years or more, while about a third of those who were lymph node negative develop distant metastasis within the same period. This warrants a robust biomarker(s), preferably gene expression based. In order to elucidate gene-based biomarkers for prognosis of breast cancers, gene expression profiling of primary tumors and follow-up for over 5 years has been performed. The analysis revealed a network of genes centered around the tripartite motif-containing protein 28 as an important indicator of disease progression. Short hairpin RNA–mediated knockdown of tripartite motif-containing protein 28 in breast cancer cells revealed a decreased expression of epithelial-to-mesenchymal transition markers and increased expression of epithelial markers, decreased migration and invasion, and increased chemosensitivity to doxorubicin, 5-fluorouracil, and methotrexate. Furthermore, knockdown of tripartite motif-containing protein 28 resulted in the decrease of stemness as revealed by sphere formation assay as well as decreased expression of CD44 and Bmi1. Moreover, tripartite motif-containing protein 28 knockdown significantly reduced the tumor size and lung metastasis in orthotopic tumor xenograft assay in immunocompromised mice. The tumor size was further reduced when these mice were treated with doxorubicin. These data provide evidence for tripartite motif-containing protein 28 as a biomarker and a potential therapeutic target for breast cancer.

Biologic Basis of Personalized Therapy in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer worldwide, with more than 500,000 new cases each year [1]. HNSCCs are associated with a 5-year overall survival of approximately 50 % which has remained largely unchanged [2]. Cetuximab is the only FDA-approved targeted therapy for HNSCC and there are no clinically used biomarkers for HNSCC. Personalized therapy, treatment management customized to individual patient profile, is now being investigated with greater emphasis on the molecular profile of the patients, in addition to their clinical and pathological status. With the stagnant 5-year overall survival, this shift toward molecular integration is definitely warranted. Molecular profiling has the ability to categorize the patients based on effective treatment modality, treatment response and susceptibility to develop metastasis, thereby delineating the prognosis more accurately. Response assessment further enables selection of drugs based on the status of the targeted pathways, hence ensuring better treatment outcome. Nevertheless, the success of this approach is dependent on the selection of appropriate biomarkers. Differences in the biology of cancers in terms of characteristics such as site, tissue of origin, and etiology make it mandatory to catalog the changes that are specific to each cancer as a step toward the personalized medicine approach. The advent of high-throughput techniques has enabled a global view of the molecular changes that occur at every level and hence are arguably the best option to understand and identify the probable clinically relevant and targetable pathways in HNSCC.

Analysis of solid tumor mutation profiles in liquid biopsy

Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for prognosis, treatment decisions, and disease monitoring. Matched tumor‐plasma samples were collected from 180 patients across different cancers with >90% of the samples below Stage IIIB. Tumors were profiled using next‐generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Tumor‐plasma concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Interestingly, the overall survival outcomes correlated to presurgical/at‐biopsy ctDNA levels. Baseline ctDNA stratified patients into three categories: (a) high ctDNA correlated with poor survival outcome, (b) undetectable ctDNA with good outcome, and (c) low ctDNA whose outcome was ambiguous. ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages.

Incidental Findings in Male Breast Carcinoma: A Genetic Counseling Approach

Abstract Male breast carcinoma (MBC) is a rare cancer type that accounts 1 percent of the total breast cancer cases. This substantially invites diagnosis challenges and social burdens where the individual needs more personalized therapies and genetic counseling to cope with the condition. This study aims to analyse a total of four male breast cancer cases with or without secondary recurrence. Tissue or saliva samples were analysed with informed and written consent for each individual subjecting to next generation sequencing aiming high throughput investigations. The results revealed two cancer syndromes in an individual with breast and thyroid carcinoma and mutations in PI3KCA, PTEN, NBN, RB1 genes. The rest three cases were identified with alterations in NBN, BRIP1 and BRCA2 mutations. Genetic counseling was provided to each participant and the responses were noted upon post-test targeted therapies.

Genotype-Phenotype Analysis in an Indian Family Affected with Li-Fraumeni Syndrome-Role of Genetic Counselling

Abstract Li-Fraumeni syndrome (LFS) is associated with the high risk of a diverse spectrum of childhood and adult-onset malignancies with a predominance of the soft-tissue sarcomas, osteosarcoma, breast cancer, brain tumor, adrenocortical carcinomas, Wilms tumor, leukaemia and several other LFS-associated cancer types. This paper reports a case of a 43 years male diagnosed with an undifferentiated, high grade sarcoma. Genetic testing by Next Generation Sequencing revealed a heterozygous likely novel pathogenic germline mutation in the TP53 gene (c.323delG; p.Gly108ValfsTer15) in the proband. Post-test genetic counselling referred the family screening and the other eight family members were found to be carrier for the same variant. Thus the researchers have tried to describe the genotype-phenotype correlation for the LFS with the TP53 mutation which may have contributed to the variable phenotypes in the reported family with reduced/ incomplete penetrance. In this paper the researchers have also tried to highlight the cancer genetic counselling to detect an inherited cancer syndrome