Publications by authors named "Dhanya K Nambiar"

12 Publications

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Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer.

Br J Cancer 2021 Feb 3;124(3):604-615. Epub 2020 Nov 3.

Department of Radiation Oncology, Stanford University School of Medicine, Palo Alto, CA, 94304, USA.

Background: To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment.

Methods: Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models.

Results: We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis.

Conclusions: SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.
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http://dx.doi.org/10.1038/s41416-020-01137-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851402PMC
February 2021

The role of Glial cell derived neurotrophic factor in head and neck cancer.

PLoS One 2020 21;15(2):e0229311. Epub 2020 Feb 21.

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, United States of America.

Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRα1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229311PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034888PMC
May 2020

Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance.

J Clin Invest 2019 12;129(12):5553-5567

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA.

Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.
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http://dx.doi.org/10.1172/JCI129025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877340PMC
December 2019

The Immune Subtypes and Landscape of Squamous Cell Carcinoma.

Clin Cancer Res 2019 06 4;25(12):3528-3537. Epub 2019 Mar 4.

Department of Radiation Oncology, Stanford University School of Medicine, Palo Alto, California.

Purpose: To identify immune subtypes and investigate the immune landscape of squamous cell carcinomas (SCC), which share common etiology and histologic features.

Experimental Design: Based on the immune gene expression profiles of 1,368 patients with SCC in the Cancer Genome Atlas (TCGA), we used consensus clustering to identify robust clusters of patients and assessed their reproducibility in an independent pan-SCC cohort of 938 patients. We further applied graph structure learning-based dimensionality reduction to the immune profiles to visualize the distribution of individual patients.

Results: We identified and independently validated six reproducible immune subtypes associated with distinct molecular characteristics and clinical outcomes. An immune-cold subtype had the least amount of lymphocyte infiltration and a high level of aneuploidy, and these patients had the worst prognosis. By contrast, an immune-hot subtype demonstrated the highest infiltration of CD8 T cells, activated NK cells, and elevated IFNγ response. Accordingly, these patients had the best prognosis. A third subtype was dominated by M2-polarized macrophages with potent immune-suppressive factors such as TGFβ signaling and reactive stroma, and these patients had relatively inferior prognosis. Other subtypes showed more diverse immunologic features with intermediate prognoses. Finally, our analysis revealed a complex immune landscape consisting of both discrete clusters and continuous spectrum.

Conclusions: This study provides a conceptual framework to understand the tumor immune microenvironment of SCCs. Future work is needed to evaluate its relevance in the design of combination treatment strategies and guiding optimal selection of patients for immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-4085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571041PMC
June 2019

Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes.

Proc Natl Acad Sci U S A 2018 06 24;115(24):6279-6284. Epub 2018 May 24.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305;

Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.
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http://dx.doi.org/10.1073/pnas.1802184115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004437PMC
June 2018

Chemical Space Mimicry for Drug Discovery.

J Chem Inf Model 2017 04 3;57(4):875-882. Epub 2017 Apr 3.

Department of Radiation Oncology, Stanford University School of Medicine , Stanford, California 94305, United States.

We describe a new library generation method, Machine-based Identification of Molecules Inside Characterized Space (MIMICS), that generates sets of molecules inspired by a text-based input. MIMICS-generated libraries were found to preserve distributions of properties while simultaneously increasing structural diversity. Newly identified MIMICS-generated compounds were found to be bioactive as inhibitors of specific components of the unfolded protein response (UPR) and the VEGFR2 pathway in cell-based assays, thus confirming the applicability of this methodology toward drug design applications. Wider application of MIMICS could facilitate the efficient utilization of chemical space.
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http://dx.doi.org/10.1021/acs.jcim.6b00754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802964PMC
April 2017

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

Mol Carcinog 2017 03 5;56(3):833-848. Epub 2016 Sep 5.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado.

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/mc.22537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637733PMC
March 2017

Angiogenesis Assays.

Methods Mol Biol 2016 ;1379:107-15

Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.

Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.
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http://dx.doi.org/10.1007/978-1-4939-3191-0_10DOI Listing
September 2016

Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

Mol Cancer Ther 2015 Dec 29;14(12):2722-34. Epub 2015 Oct 29.

Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. School of Life Sciences, Central University of Gujarat, Gandhinagar, India.

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674330PMC
December 2015

Hypoxia induces triglycerides accumulation in prostate cancer cells and extracellular vesicles supporting growth and invasiveness following reoxygenation.

Oncotarget 2015 Sep;6(26):22836-56

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado, USA.

Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA.
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http://dx.doi.org/10.18632/oncotarget.4479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673203PMC
September 2015

Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells.

Biochem Biophys Res Commun 2015 Jan 24;456(1):262-8. Epub 2014 Nov 24.

Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India; School of Life Sciences, Central University of Gujarat, Gandhinagar, India. Electronic address:

Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p<0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro-angiogenic (VEGF, iNOS), migratory (MMP-2) and EMT promoting proteins (uPA, vimentin, N-cadherin) were up-regulated by IR in PCa cells. Interestingly, all of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR-activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3, by silibinin.
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http://dx.doi.org/10.1016/j.bbrc.2014.11.069DOI Listing
January 2015

Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1.

Oncotarget 2014 Oct;5(20):10017-33

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. University of Colorado Cancer Center, Aurora, CO, USA.

Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259402PMC
http://dx.doi.org/10.18632/oncotarget.2488DOI Listing
October 2014