Browsing by Author "Nakshatri, Harikrishna"
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Item A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle(Elsevier, 2023-03-31) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Murphy, Michael P.; Wanczyk, Kristen E.; Simpson, Edward; Chen, Duojiao; Gao, Hongyu; Liu, Yunlong; Doud, Emma H.; Mosley, Amber L.; Nakshatri, Harikrishna; Surgery, School of MedicineSkeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.Item A triple hormone receptor ER, AR, and VDR signature is a robust prognosis predictor in breast cancer(Springer Nature, 2024-09-13) Omar, Mohamed; Harrell, J. Chuck; Tamimi, Rulla; Marchionni, Luigi; Erdogan, Cihat; Nakshatri, Harikrishna; Ince, Tan A.; Surgery, School of MedicineBackground: Despite evidence indicating the dominance of cell-of-origin signatures in molecular tumor patterns, translating these genome-wide patterns into actionable insights has been challenging. This study introduces breast cancer cell-of-origin signatures that offer significant prognostic value across all breast cancer subtypes and various clinical cohorts, compared to previously developed genomic signatures. Methods: We previously reported that triple hormone receptor (THR) co-expression patterns of androgen (AR), estrogen (ER), and vitamin D (VDR) receptors are maintained at the protein level in human breast cancers. Here, we developed corresponding mRNA signatures (THR-50 and THR-70) based on these patterns to categorize breast tumors by their THR expression levels. The THR mRNA signatures were evaluated across 56 breast cancer datasets (5040 patients) using Kaplan-Meier survival analysis, Cox proportional hazard regression, and unsupervised clustering. Results: The THR signatures effectively predict both overall and progression-free survival across all evaluated datasets, independent of subtype, grade, or treatment status, suggesting improvement over existing prognostic signatures. Furthermore, they delineate three distinct ER-positive breast cancer subtypes with significant survival in differences-expanding on the conventional two subtypes. Additionally, coupling THR-70 with an immune signature identifies a predominantly ER-negative breast cancer subgroup with a highly favorable prognosis, comparable to ER-positive cases, as well as an ER-negative subgroup with notably poor outcome, characterized by a 15-fold shorter survival. Conclusions: The THR cell-of-origin signature introduces a novel dimension to breast cancer biology, potentially serving as a robust foundation for integrating additional prognostic biomarkers. These signatures offer utility as a prognostic index for stratifying existing breast cancer subtypes and for de novo classification of breast cancer cases. Moreover, THR signatures may also hold promise in predicting hormone treatment responses targeting AR and/or VDR.Item Aberrant epigenetic and transcriptional events associated with breast cancer risk(BMC, 2022-02-09) Marino, Natascia; German, Rana; Podicheti, Ram; Rusch, Douglas B.; Rockey, Pam; Huang, Jie; Sandusky, George E.; Temm, Constance J.; Althouse, Sandra; Nephew, Kenneth P.; Nakshatri, Harikrishna; Liu, Jun; Vode, Ashley; Cao, Sha; Storniolo, Anna Maria V.; Medicine, School of MedicineBackground: Genome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N = 146, median age = 39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina's HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation. Results: Transcriptomic analysis identified 69 differentially expressed genes between women at high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR < 0.05 and fold change ≥ 2. Majority of the identified genes were involved in DNA damage checkpoint, cell cycle, and cell adhesion. Two genes, FAM83A and NEK2, were overexpressed in tissue sections (FDR < 0.01) and primary epithelial cells (p < 0.05) from high-risk breasts. Moreover, 1698 DNA methylation changes were identified in high-risk breast tissues (FDR < 0.05), partially overlapped with cancer-related signatures, and correlated with transcriptional changes (p < 0.05, r ≤ 0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified. Conclusions: Normal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues, and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.Item Acquisition, processing, and single-cell analysis of normal human breast tissues from a biobank(Cell Press, 2021-12-16) Bhat-Nakshatri, Poornima; Marino, Natascia; Gao, Hongyu; Liu, Yunlong; Storniolo, Anna Maria; Nakshatri, Harikrishna; Surgery, School of MedicineThe Komen Tissue Bank is the only biorepository in the world for normal breast tissues from women. Below we report the acquisition and processing of breast tissue from volunteer donors and describe an experimental and analysis pipeline to generate a single-cell atlas. This atlas is based on single-cell RNA-seq and is useful to derive breast epithelial cell subcluster-specific gene expression signatures, which can be applied to breast cancer gene expression data to identify putative cell-of-origin. For complete details on the use and execution of this protocol, please refer to Bhat-Nakshatri et al. (2021).Item Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype(Wiley, 2021-11) Bahcecioglu, Gokhan; Yue, Xiaoshan; Howe, Erin; Guldner, Ian; Stack, M. Sharon; Nakshatri, Harikrishna; Zhang, Siyuan; Zorlutuna, Pinar; Surgery, School of MedicineAge is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.Item Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model(Wiley, 2021) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Prasad, Mayuri S.; Jacobsen, Max H.; Ovalle, Gabriela; Maguire, Calli; Sandusky, George; Trivedi, Trupti; Mohammad, Khalid S.; Guise, Theresa; Penthala, Narsimha R.; Crooks, Peter A.; Liu, Jianguo; Zimmers, Teresa; Nakshatri, Harikrishna; Surgery, School of MedicineBackground: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. Methods: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. Results: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1β, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. Conclusions: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.Item AKT Alters Genome-Wide Estrogen Receptor α Binding and Impacts Estrogen Signaling in Breast Cancer(American Society for Microbiology, 2008-12) Bhat-Nakshatri, Poornima; Wang, Guohua; Appaiah, Hitesh; Luktuke, Nikhil; Carroll, Jason S.; Geistlinger, Tim R.; Brown, Myles; Badve, Sunil; Liu, Yunlong; Nakshatri, HarikrishnaEstrogen regulates several biological processes through estrogen receptor α (ERα) and ERβ. ERα-estrogen signaling is additionally controlled by extracellular signal activated kinases such as AKT. In this study, we analyzed the effect of AKT on genome-wide ERα binding in MCF-7 breast cancer cells. Parental and AKT-overexpressing cells displayed 4,349 and 4,359 ERα binding sites, respectively, with ∼60% overlap. In both cell types, ∼40% of estrogen-regulated genes associate with ERα binding sites; a similar percentage of estrogen-regulated genes are differentially expressed in two cell types. Based on pathway analysis, these differentially estrogen-regulated genes are linked to transforming growth factor β (TGF-β), NF-κB, and E2F pathways. Consistent with this, the two cell types responded differently to TGF-β treatment: parental cells, but not AKT-overexpressing cells, required estrogen to overcome growth inhibition. Combining the ERα DNA-binding pattern with gene expression data from primary tumors revealed specific effects of AKT on ERα binding and estrogen-regulated expression of genes that define prognostic subgroups and tamoxifen sensitivity of ERα-positive breast cancer. These results suggest a unique role of AKT in modulating estrogen signaling in ERα-positive breast cancers and highlights how extracellular signal activated kinases can change the landscape of transcription factor binding to the genome.Item AKT1 Transcriptomic Landscape in Breast Cancer Cells(MDPI, 2022-07-25) George, Bijesh; Gui, Bin; Raguraman, Rajeswari; Paul, Aswathy Mary; Nakshatri, Harikrishna; Pillai, Madhavan Radhakrishna; Kumar, Rakesh; Surgery, School of MedicineOverexpression and hyperactivation of the serine/threonine protein kinase B (AKT) pathway is one of the most common cellular events in breast cancer progression. However, the nature of AKT1-specific genome-wide transcriptomic alterations in breast cancer cells and breast cancer remains unknown to this point. Here, we delineate the impact of selective AKT1 knock down using gene-specific siRNAs or inhibiting the AKT activity with a pan-AKT inhibitor VIII on the nature of transcriptomic changes in breast cancer cells using the genome-wide RNA-sequencing analysis. We found that changes in the cellular levels of AKT1 lead to changes in the levels of a set of differentially expressed genes and, in turn, imply resulting AKT1 cellular functions. In addition to an expected positive relationship between the status of AKT1 and co-expressed cellular genes, our study unexpectedly discovered an inherent role of AKT1 in inhibiting the expression of a subset of genes in both unstimulated and growth factor stimulated breast cancer cells. We found that depletion of AKT1 leads to upregulation of a subset of genes-many of which are also found to be downregulated in breast tumors with elevated high AKT1 as well as upregulated in breast tumors with no detectable AKT expression. Representative experimental validation studies in two breast cancer cell lines showed a reasonable concurrence between the expression data from the RNA-sequencing and qRT-PCR or data from ex vivo inhibition of AKT1 activity in cancer patient-derived cells. In brief, findings presented here provide a resource for further understanding of AKT1-dependent modulation of gene expression in breast cancer cells and broaden the scope and significance of AKT1 targets and their functions.Item Antitumor Properties of Novel Sesquiterpene Lactone Analogs as NFκB Inhibitors that Bind to the IKKβ Ubiquitin-like Domain (ULD)(Elsevier, 2021) Penthala, Narsimha R.; Balasubramaniam, Meenakshisundaram; Dachavaram, Soma Shekar; Morris, Earl J.; Bhat-Nakshatri, Poornima; Ponder, Jessica; Jordan, Craig T.; Nakshatri, Harikrishna; Crooks, Peter A.; Surgery, School of MedicineMelampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKβ subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and in vitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values <2 μM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231 cells, but did inhibit phophorylation of both p65 and IκBα in SUM149 cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149 cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKβ subunit. These data suggest that in SUM149 cells, 7b is likely acting as an allosteric inhibitor of IKKβ, whereas in M9 ENL AML and OV-MD-231 cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKβ that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.Item ANTXR1, a stem cell enriched functional biomarker, connects collagen signaling to cancer stem-like cells and metastasis in breast cancer(American Association for Cancer Research, 2013-09-15) Chen, Daohong; Bhat-Nakshatri, Poornima; Goswami, Chirayu; Badve, Sunil; Nakshatri, HarikrishnaCancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor ANTXR1 has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f+/EpCAM−/Lin−) expressed higher levels of ANTXR1 compared to mature luminal cells. CD49f+/EpCAM−, CD44+/EpCAM−, CD44+/CD24− or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44+/CD24−/ANTXR1+ cells displayed enhanced self-renewal as measured by mammosphere assay compared to CD44+/CD24−/ANTXR1− cells. Activation of ANTXR1 by its natural ligand C5A, a fragment of collagen VI α3, increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor LRP6, phosphorylation of GSK3α/β and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression, self-renewal, invasion, tumorigenicity and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A, which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Further, ANTXR1 is among the 10% most overexpressed genes in breast cancer and is co-expressed with collagen VI. Thus, ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment, linking it to a stemness signaling network drives metastatic progression.