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The role of human microbiota in cancer initiation and progression is recognized in recent years. In order to investigate the interactions between cancer cells and microbes, a systematic analysis using various emerging techniques is required. Owing to the label-free, non-invasive and molecular fingerprinting characteristics, vibrational spectroscopy is uniquely suited to decode and understand the relationship and interactions between cancer and the microbiota at the molecular level. In this review, we first provide a quick overview of the fundamentals of vibrational spectroscopic techniques, namely Raman and infrared spectroscopy. Next, we discuss the emerging evidence underscoring utilities of these spectroscopic techniques to study cancer or microbes separately, and share our perspective on how vibrational spectroscopy can be employed at the intersection of the two fields. Finally, we envision the potential opportunities in exploiting vibrational spectroscopy not only in basic cancer-microbiome research but also in its clinical translation, and discuss the challenges in the bench to bedside translation.

Delineating the molecular and morphological changes that cancer cells undergo in response to extracellular stimuli is crucial for identifying factors that promote tumor progression. Label-free optical imaging offers a potentially promising route for retrieving such single-cell information by generating detailed visualization of the morphology and determining alterations in biomolecular composition. The potential of such nonperturbative morphomolecular microscopy for analyzing microbiota-cancer cell interactions has been surprisingly underappreciated, despite the growing evidence of the critical role of dysbiosis in malignant transformations. Here, using a model system of breast cancer cells, we show that label-free Raman microspectroscopy and quantitative phase microscopy can detect biomolecular and morphological changes in single cells exposed to

The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of Bacteroides fragilis in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit “BFT memory” from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic Bacteroides fragilis. This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the β-catenin and Notch1 axis as its functional mediators, and proposes the concept of “BFT memory” that can have far-reaching biological implications after initial exposure to ETBF.Significance:B. fragilis is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a “BFT memory” with long-term implications, functionally mediated by the β-catenin and Notch1 pathways.This article is highlighted in the In This Issue feature, p. 995

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Related Article from Big Punches Come in Nanosizes for Chemoprevention

The human body is colonized by the microbial cells that are estimated to be as abundant as human cells, yet their genome is roughly 100 times the human genome, providing significantly more genetic diversity. The past decade has observed an explosion of interest in examining the existence of microbiota in the human body and understanding its role in various diseases including inflammatory bowel disease, neurologic diseases, cardiovascular disorders, and cancer. Many studies have demonstrated differential community composition between normal tissue and cancerous tissue, paving the way for investigations focused on deciphering the cause-and-effect relationships between specific microbes and initiation and progression of various cancers. Also, evolving are the strategies to alter tumor-associated dysbiosis and move it toward eubiosis with holistic approaches to change the entire neighborhood or to neutralize pathogenic strains. In this review, we discuss important pathogenic bacteria and the underlying mechanisms by which they affect cancer progression. We summarize key microbiota alterations observed in multiple tumor niches, their association with clinical stages, and their potential use in cancer diagnosis and management. Finally, we discuss microbiota-based therapeutic approaches.

Exploratory analysis comparing weight loss across various clinical subgroups.

Supplementary Table 1

Change in waist circumference from baseline to six-months in patients assigned to POWER-remote and self-directed arms.

Purpose:We initiated a clinical trial to determine the proportion of breast cancer survivors achieving ≥5% weight loss using a remotely delivered weight loss intervention (POWER-remote) or a self-directed approach, and to determine the effects of the intervention on biomarkers of cancer risk including metabolism, inflammation, and telomere length.Experimental Design:Women with stage 0–III breast cancer, who completed local therapy and chemotherapy, with a body mass index ≥25 kg/m2 were randomized to a 12-month intervention (POWER-remote) versus a self-directed approach. The primary objective was to determine the number of women who achieved at least 5% weight loss at 6 months. We assessed baseline and 6-month change in a panel of adipocytokines (adiponectin, leptin, resistin, HGF, NGF, PAI1, TNFα, MCP1, IL1β, IL6, and IL8), metabolic factors (insulin, glucose, lipids, hs-CRP), and telomere length in peripheral blood mononuclear cells.Results:From 2013 to 2015, 96 women were enrolled, and 87 were evaluable for the primary analysis; 45 to POWER-remote and 42 to self-directed. At 6 months, 51% of women randomized to POWER-remote lost ≥5% of their baseline body weight, compared with 12% in the self-directed arm [OR, 7.9; 95% confidence interval (CI), 2.6–23.9; P = 0.0003]; proportion were similar at 12 months (51% vs 17%, respectively, P = 0.003). Weight loss correlated with significant decreases in leptin, and favorable modulation of inflammatory cytokines and lipid profiles. There was no significant change in telomere length at 6 months.Conclusions:A remotely delivered weight loss intervention resulted in significant weight loss in breast cancer survivors, and favorable effects on several biomarkers.

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PDF file - 131KB, Supplemental Figure 1 Withaferin A inhibits cell viability of breast cancer cells.

Supplementary Figure Legends 1-2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Supplementary Figure 1 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

PDF file - 141KB, Supplemental Figure 6 ERK and RSK activation is important for WFA-mediated inhibition of clonogenicity of breast cancer cells.

Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels that are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK (ribosomal S6 kinase) in breast cancer cells. Pursuing this observation, we defined activation of extracellular signal–regulated kinase (ERK)–RSK and ETS-like transcription factor 1 (Elk1)–CHOP (C-EBP homologous protein) kinase pathways in upregulating transcription of the death receptor 5 (DR5). Through this route, WFA acted as an effective DR5 activator capable of potentiating the biologic effects of celecoxib, etoposide, and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and mouse mammary tumor virus (MMTV)-neu mouse models in a manner associated with activation of the ERK/RSK axis, DR5 upregulation, and elevated nuclear accumulation of Elk1 and CHOP. Together, our results offer mechanistic insight into how WFA inhibits breast tumor growth. Cancer Res; 74(9); 2617–29. ©2014 AACR.

Supplementary Figure 2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

PDF file - 99KB, Supplementary Figure 8 CHOP is important for Withaferin A mediated inhibition of breast cancer cell growth.

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PDF file - 115KB, Supplemental Figure 3 WFA increases phosphorylation of ERK and RSK.

PDF file - 147KB, Supplemental Figure 4 WFA activates RSK in an ERK-dependent manner.

PDF file - 126KB, Supplemental Figure 7 Withaferin A induces phosphorylation of Elk1 in ERK-dependent manner.

PDF file - 156KB, Supplementary Figure 9 WFA increases DR5 expression in multiple breast cancer cell lines and CHOP and Elk1 contribute to WFA-induced upregulation of Death receptor 5 in breast cancer cells.

PDF file - 112KB, Supplemental Figure 5 WFA activates RSK in an ERK-dependent manner. ERK and RSK activation is important for WFA-mediated cleavage of apoptotic marker, PARP.

PDF file - 89KB, Supplemental Figure 2 Withaferin A treatment inhibits expression of anti-apoptotic proteins, Survivin and XIAP and increases PARP cleavage.

It is felt that Modern Banking has become wholly customer – driven and technology driven. During the last decade, technology has been dramatically transforming the banking activities in India. Driven by challenges on competition, rising customer expectation and shrinking margins, banks have been using technology to reduce cost. Apart from competitive environment, there has been deregulation as to rate of interest, technology intensive delivery channel like Internet Banking, Tele Banking, Mobile banking and Automated Teller Machines (ATMs) etc. have created a multiple choice to user of the bank. The banking business is becoming more and more complex with the changes emanating from the liberalization and globalization. For a new bank, customer creation is important, but an established bank it is the retention is much more efficient and cost effective mechanism. Customer Relationship Management (CRM) would also make Indian bankers realize that the purpose of their business is to create and keep a customer and to view the entire business process as consisting of Highly Integrated effort to discover, create and satisfy customer needs. But it is surprising to note that much of the activities of the banking and financial remain focused on customer creation not retention.

Background: African American breast cancer patients (AA) are diagnosed at a younger age and present more frequently with triple-negative/Basal tumors than Caucasian American patients (CA). High prevalence of obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome in AA may confound attempts to evaluate the influence of race on gene expression. Previously we showed that differentially expressed genes (DEGs) between AA and CA within the Basal subtype were related to metabolism, translation, and cell signaling pathways (Nunes et al. 2019). However, AA had higher obesity and T2DM rates than CA, and we were unable to distinguish between the influence of metabolic factors and race. In the current analysis, we aim to dissect these factors by comparing clinical and molecular features of Basal-type breast tumors in obese AA and CA. Methods: The prospective, observational FLEX Registry (NCT03053193) includes stage I-III breast cancer patients who receive 70-gene signature (MammaPrint, MP)/80-gene signature (BluePrint, BP) testing and consent to full transcriptome and clinical data collection. This interim substudy included 50 AA and 96 CA (n=146), enrolled from 2017 to present, all obese by body mass index (BMI, ≥30) and whose tumors were MP High Risk and BP Basal subtype. AA were significantly younger (mean, 55 years) than CA (mean, 60 years, p=0.02); thus, an age distribution-matched subset (n=49 AA, n=49 CA) was added for comparison. Gene expression data were quantile normalized using R limma package; DEGs were compared between groups in the following: (1) all AA (n=50) and CA (n=96), (2) AA and 3 random selections of CA (n=50 pairs), and (3) age-matched AA and CA (n=49 pairs). Results: Clinical factors, including tumor stage, nodal stage, and T2DM status were similar between AA and CA, regardless of age-matching. Most tumors were T1/2 (83% AA, 88% CA) and negative for nodal involvement (77% AA, 68% CA). 94% of tumors from AA and 74% of tumors from CA were grade 3 (p=0.17). Notably, 32% of tumors from AA and 46% of tumors from CA were ER+ by immunohistochemistry. Age-matched AA and CA had a 20% rate of T2DM. 152 DEGs were significant (adjusted p Conclusions: Higher prevalence of obesity/T2DM in AA has been proposed as a key factor to explain racial disparities in breast cancer incidence and prognosis, but the current results suggest that race may influence DEGs more than differences in tumor subtype, age, or metabolic factors. This comparison also emphasizes the importance of matched clinical features for DEG analysis and suggests disparities in AA beyond those attributable to clinical differences within the population. DEGs in AA suggest upregulation of Notch-associated aggressiveness, which may be particularly relevant under hypoxic conditions (e.g., obesity), and pathways associated with stemness, metastasis, and chemotherapy resistance. Notch pathway also interacts with key oncogenic pathways, and future studies will reveal the molecular networks underlying racial disparity in AA and CA breast cancer patients. Citation Format: Dipali Sharma, Lisa E. Blumencranz, Heather M. Kling, Sahra Uygun, Sarah Untch, William Audeh, Jennifer A. Crozier, Mehran Habibi, Raquel Nunes. Racial disparities within basal-type breast cancer: Clinical and molecular features of African American and Caucasian obese patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-68.

Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to White American (WA) women (5-year relative survival of only 14% for AA in comparison to 36% for WA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than WA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to WA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to WA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and WA TNBC cell lines to query the status self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in WA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to WA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to WA-TNBC cells. We observed that GLI1 and NICD co-localize and co-immunoprecipitate in AA-TNBC indicating a direct interaction between these two transcription factors. High expression of GLI1 and Notch1 correlated with poor recurrence free survival in TNBC patients. Analyses of clinical samples revealed higher levels of nuclear NICD and GLI1 in AA-TNBC in comparison to WA-TNBC. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC tumor growth in mice. Also, ex vivo analyses of tumor cells showed reduced migration and invasion potential as well as downregulation of CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI1 and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Our studies propose a ‘triple drug regimen’ for AA-TNBC tumors based on its molecular circuitry. Citation Format: Sumit Siddharth, Sheetal Parida, Nethaji Muniraj, Shawn M Hercules, Arumugam Nagalingam, Balázs Győrffy, Juliet Daniel, Dipali Sharma. Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-86.

Hepatocellular carcinoma (HCC) incidence, as well as related mortality, has been steadily increasing in the USA and across the globe, partly due to the lack of effective therapeutic options for advanced HCC. Though sorafenib is considered standard-of-care for advanced HCC, it only improves median survival by a few months when compared to placebo. Sorafenib is also associated with several unpleasant side effects that often lead to early abatement of therapy. Here, we investigate whether a combination regimen including low-dose sorafenib and a non-toxic dose of anti-diabetic drug metformin can achieve effective inhibition of HCC. Indeed, combining metformin with low-dose sorafenib inhibited growth, proliferation, migration, and invasion potential of HCC cells. We observed a 5.3- and 1.9-fold increase in sub-G1 population in the combination treatment compared to sorafenib alone. We found that the combination of metformin enhanced the efficacy of sorafenib and inhibited the MAPK/ERK/Stat3 axis. Our in vivo studies corroborated the in vitro findings, and mice harboring HepG2-derived tumors showed effective tumor reduction upon treatment with low-dose sorafenib and metformin combination. This work sheds light on a therapeutic strategy aiming to augment sorafenib efficacy or dose-de-escalation that may prove beneficial in circumventing sorafenib resistance as well as minimizing related side effects.

Background: 41.9% of adult US population is obese and every 5-unit BMI increase is associated with 12 percent increase in breast cancer risk; it is an independent predictor of poor outcomes in breast cancer. Microbiome has emerged as a central regulator of obesity and we recently discovered that microbes can not only potentiate breast cancer growth and metastasis but also affect therapeutic response. We showed weight-loss by Diet/Exercise/Behavioral intervention (POWER-remote trial) exerts favorable effects. Here, we examine molecular underpinnings of poor therapy response in obese state and explore the gut microbiome to mitigate the same. Methods: We co-implanted lineage determined human White preAdipocytes (hWAT) with MCF7 breast cancer cells in the mammary glands of mice. Changes in tumor microenvironment and tumor cells were evaluated ex situ by RNA sequencing, flow cytometry, Raman spectroscopy, Quantitative phase microscopy, PCR, immunoblotting and functional assays. To inspect the gut microbiome, 16 obese breast cancer patients were subjected to weight loss intervention over 14 weeks. Gut microbiome was analyzed at baseline and post-intervention by microbial shotgun sequencing. Patients were divided into responders and non-responders based on % weight-loss. Results: We found that human breast cancer cells co-implanted with hWAT formed more aggressive tumors compared to controls. RNA sequencing revealed significant upregulation of multiple oncogenic pathways along with robust innate immune response. Tumor dissociated cells from hWAT-MCF7 tumors showed extensive molecular reprogramming rendering them more proliferative, motile and stem-like along with dramatic physiological changes like increased intercellular lipid accumulation. hWAT-MCF7 cells attained resistance to multiple drugs including Tamoxifen, Fulvestrant, Letrozole and Palbociclib. Simultaneous targeting of multiple signaling pathways using pharmacological inhibitors is infeasible in clinical setting hence we explored the obesity-microbiome axis. Obese breast cancer patients underwent POWER-remote intervention and their gut microbiome analysis showed significant differential expression of many microbes. Based on abundance and metabolite profile, F. prausnitzii was selected as the candidate of interest. On treatment with F. prausnitzii growth media, acquired multi-drug resistance in hWAT-MCF7 cells could be reversed suggesting that increase in gut population of the bacterium could be helpful in sensitizing patients unresponsive to therapy. Conclusion: Direct interaction between breast cancer cells and WAT rendered them resistant to multiple drugs. Metabolites produced by weight-loss-associated bacterium F. prausnitzii reversed adiposity-induced multi-drug resistance, hence, can potentially have therapeutic benefits in obese breast cancer patients. Citation Format: Sheetal Parida, Sumit Siddharth, Nethaji Muniraj, Zhenhui Liu, Ishan Barman, Michael Smith, Jenni Sheng, Vered Stearns, Janelle W. Coughlin, Dipali Sharma. Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5916.

Disparate occurrence of breast cancer remains an intriguing question since only a subset of women with known risk factors develop cancer. Recent studies suggest an active role of local and distant microbiota in breast cancer initiation, progression, and overall prognosis. A dysbiotic microbiota predisposes the body to develop cancer by inducing genetic instability, initiating DNA damage and proliferation of the damaged progeny, eliciting favorable immune response, metabolic dysregulation and altered response to therapy. In this review, we present our analyses of the existing datasets and discuss the local dysbiosis observed in breast cancer patients and different aspects of breast carcinogenesis that can be potentially influenced by local breast microbiota. Striking differences between microbial community compositions in breast of cancer patients compared to healthy individuals were noted. Differences in microbiome were also apparent between benign and malignant disease and between nipple aspirate fluid of healthy individuals and breast survivors. We also discuss the identification of distinct bacterial, fungal, viral as well as parasite signatures for breast cancer. These microbes are capable of producing numerous secondary metabolites that can act as signaling mediators effecting breast cancer progression. We review how microbes potentially alter response to therapy affecting drug metabolism, pharmacokinetics, anti-tumor effects and toxicity. In conclusion, breast harbors a community of microbes that can communicate with the host cells inducing downstream signaling pathways and modulating various aspects of breast cancer growth and metastatic progression and an improved understanding of microbial dysbiosis can potentially reduce breast cancer risk and improve outcomes of breast cancer patients. The human microbiome, now referred to as, the "forgotten organ" contains a metagenome that is 100-fold more diverse compared to the human genome, thereby, is critically associated with human health [1,2]. With the revelations of the human microbiome project and advent of deep sequencing techniques, a plethora of information has been acquired in recent years. Body sites like stomach, bladder and lungs, once thought to be sterile, are now known to harbor millions of indigenous microbial species. Approximately 80% of the healthy microbiome consists of Firmicutes and Bacteroidetes accompanied by Verrucomicrobia, Actinobacteria, Proteobacteria, Tenericutes and Cyanobacteria [2-7]. The role of microbiome in diabetes, obesity and even neurodegenerative diseases was greatly appreciated in the last decade [1,7-14] and now it has been established that microbiome significantly contributes to many organ specific cancers [1,15,16].