Integrated genomics approach to identify biologically relevant alterations in fewer samples

Chandrani, Pratik; Upadhyay, Pawan; Iyer, Prajish; Tanna, Mayur; Shetty, Madhur; Raghuram, Gorantala Venkata; Oak, Ninad; Singh, Ankita; Chaubal, Rohan; Ramteke, Manoj P.; Gupta, Sudeep; Dutt, Amit

doi:10.1186/s12864-015-2138-4

Cited by 10 publications

(11 citation statements)

References 67 publications

(79 reference statements)

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“…Activated NOTCH1 form more number of spheres as compared to vector control cells post 5 days (Supplementary Figure S6, S6C). However, given that AW13516 cells are HPV negative [25] and harbor wild-type p16INK4A and mutant Tp53 [23], ectopic expression of full-length NOTCH1 or NICD led to continuous cell death and senescence mediated growth arrest (Supplementary Figure S6, S6G), as described earlier [26-28]. …”

Section: Resultsmentioning

confidence: 64%

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Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

et al. 2016

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BackgroundNotch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC).Patients and MethodsWe analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays.ResultsWe show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029).ConclusionWe anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer.

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Section: Resultsmentioning

confidence: 64%

“…First, we checked for the presence of Notch pathway transcript expression by real-time PCR and western analysis of NOTCH1 protein using multiple head and neck cancers cell lines (NT8e, AW13516, CAL27 and DOK) [23]. NT8e and CAL27 cells showed higher expression of NOTCH1 as compared to AW13516 and DOK cells (Supplementary Figure S4A, S4B).…”

Section: Resultsmentioning

confidence: 99%

Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

et al. 2016

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“…Anchorage independent growth assay and immunoblotting were carried out as described earlier [17], and as detailed in the supplementary methods, available at Annals of Oncology online.…”

Section: Methodsmentioning

confidence: 99%

Drug-sensitiveFGFR3 mutations in lung adenocarcinoma

et al. 2017

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BackgroundLung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin.Materials and methodsForty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-type FGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors using in vitro and xenograft mouse models.ResultsWe present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations of FGFR3 are present in 20 of 363 (5.5%) patients. These FGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition of FGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressing FGFR3 constructs and growth of tumors driven by FGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating the in vitro findings. Interestingly, the FGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboring KRAS mutations.ConclusionWe present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicate FGFR3 as a novel therapeutic in lung adenocarcinoma.

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“…All experiments were performed in triplicates as described earlier. 34 Briefly, anchorage-independent growth was assessed for the knockdown clones of ERBB2 and EGFR along with respective scrambled control. About 1 mL of 2× DMEM supplemented with 20% FBS containing (1 mL of 1.6% agar) to obtain 0.8% agar was added to the six well plate as bottom agar and was allowed to solidify.…”

Section: Soft Agar Assaymentioning

confidence: 99%

ERBB2 and KRAS alterations mediate response to EGFR inhibitors in early stage gallbladder cancer

Iyer

Shrikhande

Ranjan

et al. 2018

Intl Journal of Cancer

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The uncommonness of gallbladder cancer in the developed world has contributed to the generally poor understanding of the disease. Our integrated analysis of whole exome sequencing, copy number alterations, immunohistochemical, and phospho-proteome array profiling indicates ERBB2 alterations in 40% early-stage rare gallbladder tumors, among an ethnically distinct population not studied before, that occurs through overexpression in 24% (n=25) and recurrent mutations in 14% tumors (n=44); along with co-occurring KRAS mutation in 7% tumors (n=44). We demonstrate that ERBB2 heterodimerize with EGFR to constitutively activate the ErbB signaling pathway in gallbladder cells. Consistent with this, treatment with ERBB2-specific, EGFR-specific shRNA or with a covalent EGFR family inhibitor Afatninb inhibits tumor-associated characteristics of the gallbladder cancer cells. Furthermore, we observe an in vivo reduction in tumor size of gallbladder xenografts in response to Afatinib is paralleled by a reduction in the amounts of phospho-ERK, in tumors harboring KRAS (G13D) mutation but not in KRAS (G12V) mutation, supporting an essential role of the ErbB pathway. In overall, besides implicating ERBB2 as an important therapeutic target under neo-adjuvant or adjuvant settings, we present the first evidence that the presence of KRAS mutations may preclude gallbladder cancer patients to respond to anti-EGFR treatment, similar to a clinical algorithm commonly practiced to opt for anti-EGFR treatment in colorectal cancer.

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Integrated genomics approach to identify biologically relevant alterations in fewer samples

Cited by 10 publications

References 67 publications

Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

Drug-sensitiveFGFR3 mutations in lung adenocarcinoma

ERBB2 and KRAS alterations mediate response to EGFR inhibitors in early stage gallbladder cancer

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