Detection of breast cancer stem cell gene mutations in circulating free DNA during the evolution of metastases.

PURPOSE: Limited knowledge exists on the detection of breast cancer stem cell (BCSC)-related mutations in circulating free DNA (cfDNA) from patients with advanced cancers. Identification of new cancer biomarkers may allow for earlier detection of disease progression and treatment strategy modifications. METHODS: We conducted a prospective study to determine the feasibility and prognostic utility of droplet digital polymerase chain reaction (ddPCR)-based BCSC gene mutation analysis of cfDNA in patients with breast cancer. RESULTS: Detection of quantitative BCSC gene mutation in cfDNA by ddPCR mirrors disease progression and thus may represent a valuable and cost-effective measure of tumor burden. We have previously shown that hematological and neurological expressed 1-like (HN1L), ribosomal protein L39 (RPL39), and myeloid leukemia factor 2 (MLF2) are novel targets for BCSC self-renewal, and targeting these genetic alterations could be useful for personalized genomic-based therapy. CONCLUSION: BCSC mutation detection in cfDNA may have important implications for diagnosis, prognosis, and serial monitoring.

Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models.

BACKGROUND: Breast cancer has been considered not highly immunogenic, and few patients benefit from current immunotherapies. However, new strategies are aimed at changing this paradigm. In the present study, we examined the in vivo activity of a humanized anti-programmed cell death protein 1 (anti-PD-1) antibody against triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tumor models. METHODS: To circumvent some of the limitations posed by the lack of appropriate animal models in preclinical studies of immunotherapies, partially human leukocyte antigen-matched TNBC PDX tumor lines from our collection, as well as human melanoma cell lines, were engrafted in humanized nonobese diabetic/severe combined immunodeficiency IL2Rγnull (hNSG) mice obtained by intravenous injection of CD34+ hematopoietic stem cells into nonlethally irradiated 3-4-week-old mice. After both PDXs and melanoma cell xenografts reached ~ 150-200 mm3, animals were treated with humanized anti-PD-1 antibody or anti-CTLA-4 and evaluated for tumor growth, survival, and potential mechanism of action. RESULTS: Human CD45+, CD20+, CD3+, CD8+, CD56+, CD68+, and CD33+ cells were readily identified in blood, spleen, and bone marrow collected from hNSG, as well as human cytokines in blood and engrafted tumors. Engraftment of TNBC PDXs in hNSG was high (~ 85%), although they grew at a slightly slower pace and conserved their ability to generate lung metastasis. Human CD45+ cells were detectable in hNSG-harbored PDXs, and consistent with clinical observations, anti-PD-1 antibody therapy resulted in both a significant reduction in tumor growth and increased survival in some of the hNSG PDX tumor lines, whereas no such effects were observed in the corresponding non-hNSG models. CONCLUSIONS: This study provides evidence associated with anti-PD-1 immunotherapy against TNBC tumors supporting the use of TNBC PDXs in humanized mice as a model to overcome some of the technical difficulties associated with the preclinical investigation of immune-based therapies.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway.

Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.

Breast cancer cells form primary tumors on ex vivo four-dimensional lung model.

BACKGROUND: Breast cancer mortality is most common in cancer in women, and there are no ex vivo models that can capture the primary growth of tumor with fidelity to the in vivo tumor growth. In this study, we grew human breast cancer cell lines in an acellular lung matrix of the ex vivo four-dimensional lung model to determine if they form primary tumor and the extent to which they mimic the histology and characteristics of the human tumors. MATERIALS AND METHODS: Rat lungs were harvested, decellularized, and placed in a bioreactor. To study the primary tumor growth, we seeded the lung via the trachea with human breast cancer cells SUM159, MCF7, or MDMB231 and perfused the pulmonary artery with oxygenated media. Lobectomies were performed and processed for hematoxylin and eosin, Ki-67, caspase-3, estrogen receptor, and progesterone receptor antibodies. RESULTS: All three cell lines grew in the ex vivo four-dimensional model and formed perfusable tumor nodules with similar histology and morphology as the primary tumors. SUM159 and MDAMB231 showed higher proliferation and apoptotic indices than MCF7. In addition, MCF7 retained its estrogen receptor and progesterone receptor positivity, whereas SUM159 and MDAMB 231 did not have any staining. CONCLUSIONS: Overall, our study showed that human breast cancer cells can be grown on the ex vivo four-dimensional lung model, which then form primary tumor nodules that mimic the morphology and histology of the original tumor.

Role of RPL39 in Metaplastic Breast Cancer.

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

The pivotal role of mammalian target of rapamycin inhibition in the treatment of patients with neuroendocrine tumors.

Significant advances have been made toward understanding the biology of neuroendocrine tumors (NET) in terms of defining prognosis and improving clinical management; however, many unmet needs remain. The treatment landscape for NET has evolved, with the approval of the targeted agents everolimus and sunitinib for the treatment of advanced pancreatic NET in 2011 followed by the approval of everolimus for the treatment of advanced nonfunctional gastrointestinal and lung NET in 2016. Mammalian target of rapamycin (mTOR) and components of the mTOR pathway play pivotal roles in NET pathogenesis. Effects of the mTOR inhibitor everolimus have been well documented in preclinical and clinical studies, both as monotherapy and combination therapy. mTOR inhibition as backbone therapy within the NET treatment landscape is a focus of continuing research, which includes evaluation of the growing armamentarium of approved and investigational agents as potential combination partners. Data evaluating the clinical benefits of agents targeting mTOR and related pathways (alone and in combination) in the treatment of patients with NET continue to increase. Many of the findings to date are encouraging.

The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs.

NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future.

Autophagy Inhibition to Increase Radiosensitization in Breast Cancer.

Currently, many breast cancer patients with localized breast cancer undergo breast-conserving therapy, consisting of local excision followed by radiation therapy. Following radiation therapy, breast cancer cells are noted to undergo induction of autophagy, development of radioresistance, and enrichment of breast cancer stem cell subpopulations. It is hypothesized that inhibition of the cytoprotective autophagy that arises following radiation therapy increases radiosensitivity and confers longer relapse-free survival by eliminating tumor-initiating breast cancer stem cells. Therefore, we reviewed the controversial role of autophagy in breast cancer tumorigenesis and progression, autophagy induction by radiotherapy, and utilization of autophagy inhibitors to increase radiosensitivity of breast cancer and to target radioresistant breast cancer stem cells.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor ß was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation.

In this study, we investigate a novel in vitro model to mimic heterogeneous breast tumors without the use of a scaffold while allowing for cell-cell and tumor-fibroblast interactions. Previous studies have shown that magnetic levitation system under conventional culturing conditions results in the formation of three-dimensional (3D) structures, closely resembling in vivo tissues (fat tissue, vasculature, etc.). Three-dimensional heterogeneous tumor models for breast cancer were designed to effectively model the influences of the tumor microenvironment on drug efficiency. Various breast cancer cells were co-cultured with fibroblasts and then magnetically levitated. Size and cell density of the resulting tumors were measured. The model was phenotypically compared to in vivo tumors and examined for the presence of ECM proteins. Lastly, the effects of tumor stroma in the 3D in vitro model on drug transport and efficiency were assessed. Our data suggest that the proposed 3D in vitro breast tumor is advantageous due to the ability to: (1) form large-sized (millimeter in diameter) breast tumor models within 24 h; (2) control tumor cell composition and density; (3) accurately mimic the in vivo tumor microenvironment; and (4) test drug efficiency in an in vitro model that is comparable to in vivo tumors.

Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44(+) /CD24(-/low) cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44(+) /CD24(-/low) CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44(+) /CD24(-/low) CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus-activated kinase 2 (Jak2)-signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1.

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

Design and in vitro evaluation of layer by layer siRNA nanovectors targeting breast tumor initiating cells.

Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44+/CD24-/EpCAM+ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs in vitro.

Dual HER2 blockade: preclinical and clinical data.

The estrogen receptor and human epidermal growth factor receptor (HER) signaling pathways are the dominant drivers of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, targeting these pathways provides the most effective therapies in appropriately selected patients. However, de novo and acquired resistance remain major obstacles to successful treatment. By increasing the understanding of the molecular mechanisms of combined HER2-targeted therapies, we aim to be better able to select patients who would respond to these treatments and understand some of the mechanisms of resistance to HER2-targeted treatments. Recent studies have demonstrated an increased effectiveness of dual targeted HER2 therapies against HER2-amplified breast cancer as compared with single blockade. These studies have resulted in the recent US Food and Drug Administration approval of the combination of taxane chemotherapy with pertuzumab and trastuzumab in the first-line metastatic setting as well as an accelerated approval in the neoadjuvant setting. Another mechanism for overcoming resistance to HER2 targeted therapies is the antibody-drug conjugate trastuzumab-emtansine, which targets the HER2 receptor conjugated to the potent antimicrotubule agent mertansine, allowing for intracellular release of the cytotoxic drug. Studies evaluating the efficacy of dual blockade with antibody-drug conjugate are currently ongoing. This article reviews recent data on different combinations of anti-HER2 treatments as well as ongoing and future research in this area.

The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: similarities and distinct actions from soy isoflavone genistein.

Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and ß-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

Trastuzumab-resistant cells rely on a HER2-PI3K-FoxO-survivin axis and are sensitive to PI3K inhibitors.

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

Selective small molecule Stat3 inhibitor reduces breast cancer tumor-initiating cells and improves recurrence free survival in a human-xenograft model.

Metastasis and disease relapse are hypothesized to result from tumor initiating cells (TICs). Previously, we have defined a CD44+/CD24-/low mammosphere-forming tumorigenic 493-gene signature in breast cancer. Stat3 was identified as a critical node in self-renewal based on an ongoing lentiviral shRNA screen being conducted in two breast cancer cell lines SUM159 and BT549. In corroborating work, targeting the SH2 domain of Stat3 with a novel small molecule decreased the percentage of cells expressing TIC markers (CD44+/CD24-/low and ALDH+) and mammosphere formation in p-Stat3 overexpressing human breast cancer xenografts in SCID-beige mice. Importantly, we observed a four-fold improvement in the 30-day recurrence-free survival relative to docetaxel alone with the addition of the Stat3 inhibitor in the chemoresistant tumor model. Thus, these findings provide a strong impetus for the development of selective Stat3 inhibitors in order to improve survival in patients with p-Stat3 overexpressing tumors.