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Supplementary Figure S1 - PDF file 343K, Functional analysis of TLR4 protein in MDA-MB-231 cells

Supplementary Figure Legends - PDF file 88K, Supplementary Figure Legends

Overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. We found that TLR4 is overexpressed in the majority of clinical breast cancer samples and in 68% of the examined breast cancer lines. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug paclitaxel. LPS is frequently used to show a tumor-promoting role of TLR4 although this bacterial component is unlikely to be found in the breast cancer environment. We reasoned that paclitaxel-dependent activation of TLR4 is more relevant to breast cancer chemoresistance that could be mediated by activation of the NF-κB pathway leading to upregulation of prosurvival genes. To test this hypothesis, we correlated TLR4 expression with resistance to paclitaxel in two modified breast cancer lines with either depleted or overexpressed TLR4 protein. Depletion of TLR4 in naturally overexpressing MDA-MB-231 cells downregulated prosurvival genes concomitant with 2- to 3-fold reduced IC50 to paclitaxel in vitro and a 6-fold decrease in recurrence rate in vivo. Conversely, TLR4 overexpression in a negative cell line HCC1806 significantly increased expression of inflammatory and prosurvival genes along with a 3-fold increase of IC50 to paclitaxel in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. Importantly, both tumor models showed that many paclitaxel-upregulated inflammatory cytokines were coinduced with their receptors suggesting that this therapy induces autocrine tumor-promoting loops. Collectively, these results show that paclitaxel not only kills tumor cells but also enhances their survival by activating TLR4 pathway. These findings suggest that blocking TLR4 could significantly improve response to paclitaxel therapy. Mol Cancer Ther; 12(8); 1676–87. ©2013 AACR.

Supplementary Tables - PDF file 134K, Supplementary Tables

mRNA isoforms

Pharmacodynamic changes (pathways)

Response markers

Extended Materials and Methods with in depth details about experimental procedures and protocols

Pharmacodynamic markers

Supplementary tables 1 and 2; Clinical information about the patients that the PDX models were established from and pathways that are regulated in the sensitive tumors after buparlisib treatment, respectively.

Response markers (pathways)

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Supplementary Figures 1-10 supporting the study Mundt et al., "Proteomic profiling of PI3K inhibitor resistance"

The effects of nab-PXL on upregulation of cytokines in MDA-MB-231 cells expressing dominant negative (DN)-TLR4 or DN-MyD88 and control cell lines (S1); The comparison of nab-PXL and docetaxel effects on the TLR4-positive human and mouse BC cells (S2); FACS analysis of spleen and bone marrow cells (S3); Effect of a blocking mouse CD11b antibody on PXL-induced tumor recruitment of myeloid cells and blood vascular densities (S4).

Legend for Supplementary Figures S1-S4.

RT-qPCR Primers (S1); Effect of TLR4 on incidence and metastatic burden in nab-PXL treated and untreated mice (S2).

Introduction The PI3K signaling pathway is frequently active in triple negative breast cancer (TNBC), but response to PI3K inhibition is still poorly understood. To gain insights in therapy response and resistance, it is important not only to assess genetic alterations of these tumors, but also their phenotypic characteristics. Furthermore, not only is the functional status of the PI3K pathway important to know, but also other signal transduction pathways that may drive tumor growth, as they may explain primary or acquired therapy resistance. Material & Method We analyzed 17 patient derived xenograft (PDX) models of TNBC with varying response to buparlisib treatment. Long-term response (˜30 days) was assessed by comparing growth rates between buparlisib and vehicle treated PDXs. For characterization, mRNA expression analysis was performed on tumor material post 3 days of vehicle or buparlisib treatment of each PDX model. Functional pathway activity was determined using computational Bayesian networks that look at mRNA levels of pathway target genes resulting from activation (Verhaegh et al, Cancer Res 2014), amongst others for the FOXO-PI3K (van Ooijen et al, Am J Pathol, in press), ER, AR, Hedgehog, TGFbeta and Wnt pathways. These computational networks were calibrated on samples with known pathway activity, and biologically validated on various healthy and diseased cell and tissue types. Results On an initial set of 6 PDX models, pathway activity clearly varied between different TNBC PDXs, and between vehicle and buparlisib treatment. ER pathway activity was low in all samples, as expected in TNBC. Two PDX models with the most growth reduction by buparlisib showed high PI3K activity, of which one based on low FOXO activity and the other on oxidative stress. The former, best responding PDX showed a clear reduction in PI3K activity (restoring FOXO activity), when comparing 3 days buparlisib to vehicle treatment. Of the two PDX models with least growth reduction, one had low PI3K activity, while the other one, carrying a PIK3CA mutation, did show high PI3K activity (low FOXO), but this remained after treatment. Other differences in pathway activity that were found, included slightly elevated AR and Wnt activity in one PDX with good response, and somewhat higher TGFbeta activity in four PDXs (good, medium and poor response). Analysis of the remaining 11 PDXs is ongoing, including other signal transduction pathways, to investigate the variation in pathway activity across the entire panel and to shed more light on the differences in tumor biology between the PDX models. Conclusion Our computational Bayesian networks measured differences in functional activity of signal transduction pathways across a collection of TNBC PDX models, which may be expected due to the large variation between TNBC tumors. Functional PI3K activity was related to growth inhibition by buparlisib treatment, and reduction of PI3K pathway activity was observed upon treatment in responding PDX models. Other pathways also showed variation across PDXs. Further clinical evaluation of our signal transduction pathway activity measurement is ongoing. RT-qPCR based analysis is available, optimized for FFPE tissue and small samples. Citation Format: Guo Z, Verhaegh W, Hoog J, Rajput S, van Strijp D, van de Stolpe A, Li S, Ma C. Elucidating the role of functional signal transduction pathway activity in sensitivity and response of triple negative breast cancer to PI3K inhibition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-18.

Summary Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8–124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media. Lay summary To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is commonly observed in triple negative breast cancer (TNBC), leading to activation of the phosphoinositide 3-kinase (PI3K) signaling to promote tumor cell growth and chemotherapy resistance. In this study, we investigated whether adding a pan-PI3K inhibitor could improve the cytotoxic effect of eribulin, a non-taxane microtubule inhibitor, in TNBC patient-derived xenograft models (PDX) with loss of PTEN, and the underlying molecular mechanisms. Three TNBC-PDX models (WHIM6, WHIM12 and WHIM21), all with loss of PTEN expression, were tested for their response to BKM120 and eribulin, alone or in combination in vivo. In addition, the effect of drug treatment on cell proliferation and cell cycle progression were also performed in vitro using a panel of TNBC cell lines, including 2 derived from PDX models. The combination of eribulin and BKM120 led to additive or synergistic anti-tumor effect in 2 of the 3 PDX models, accompanied by an enhanced mitotic arrest and apoptosis in sensitive PDX models. In addition, the combination was synergistic in reducing mammosphere formation, and markers for epithelial-mesenchymal transition (EMT). In conclusion, PI3K inhibition induces synergistic anti-tumor effect when combined with eribulin, by enhancing mitotic arrest and apoptosis, as well as, reducing the cancer stem cell population. This study provides a preclinical rationale to investigate the therapeutic potential for the combination of PI3K inhibition and eribulin in the difficult to treat TNBC. Further studies are needed to identify the biomarkers of response for target patient selection.

// Sandeep Rajput 1 , Nimmish Khera 1 , Zhanfang Guo 1 , Jeremy Hoog 1 , Shunqiang Li 1, 2 , Cynthia X. Ma 1, 2 1 Section of Medical Oncology, Division of Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA 2 Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA Correspondence to: Cynthia X. Ma, email: cma@dom.wustl.edu Keywords: dinaciclib, triple negative breast cancer, patient derived xenograft, cyclin B1, cyclin dependent kinase 9 Received: April 08, 2016 Accepted: July 10, 2016 Published: July 28, 2016 ABSTRACT Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth. Dinaciclib is a novel CDK inhibitor currently under clinical evaluation for the treatment of advanced malignancies. In this study, we demonstrated the anti-tumor activity of dinaciclib in triple negative breast cancer (TNBC) patient derived xenograft (PDX) and cell lines in vitro and in vivo . Treatment with dinaciclib induced cell cycle arrest at G2/M phase and marked apoptosis. These changes were accompanied by reduced phosphorylation of CDK1 and retinoblastoma (Rb) protein and decreased protein levels of cyclin B1, cMYC and survivin. We further demonstrated that siRNA knockdown of CDK9, the kinase subunit of positive transcription elongation factor b (P-TEFb), instead of CDK1 or CDK2, reduced the levels of cyclin B1 and MYC in TNBC cell lines. These data support the importance of CDK9, in addition to CDK1, in mediating the growth inhibitory effect of dinaciclib in TNBC. Further investigation of CDK9 as a therapeutic target in TNBC is needed.

Within the last two decades, the advancement in cellular and cancer biology research can be defined by a few foundational pillars-one of the most important ones being the advent of small-molecule inhibitors (SMIs). Due to their several advantages over contemporary cytotoxic drugs, these engineered molecules have not only shed light on a much deeper understanding of the cellular microenvironment and signal transduction pathways, they have also provided hope of achieving clinical benefits of better patient tolerability while surpassing the efficacy of chemotherapeutic agents. It is, therefore, no surprise that research and development of SMIs has witness a steady surge amongst pharmaceutical industries and research institutions. It is, however, safe to assume that the current applications of SMIs are the mere tip of a yet-inconceivable iceberg. J. Cell. Biochem. 118: 959-961, 2017. © 2016 Wiley Periodicals, Inc.

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy. Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Emerging evidence suggests that cytotoxic therapy may actually promote drug resistance and metastasis while inhibiting the growth of primary tumors. Work in preclinical models of breast cancer has shown that acquired chemoresistance to the widely used drug paclitaxel can be mediated by activation of the Toll-like receptor TLR4 in cancer cells. In this study, we determined the prometastatic effects of tumor-expressed TLR4 and paclitaxel therapy and investigated the mechanisms mediating these effects. While paclitaxel treatment was largely efficacious in inhibiting TLR4-negative tumors, it significantly increased the incidence and burden of pulmonary and lymphatic metastasis by TLR4-positive tumors. TLR4 activation by paclitaxel strongly increased the expression of inflammatory mediators, not only locally in the primary tumor microenvironment but also systemically in the blood, lymph nodes, spleen, bone marrow, and lungs. These proinflammatory changes promoted the outgrowth of Ly6C+ and Ly6G+ myeloid progenitor cells and their mobilization to tumors, where they increased blood vessel formation but not invasion of these vessels. In contrast, paclitaxel-mediated activation of TLR4-positive tumors induced de novo generation of deep intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These results suggest that paclitaxel therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis, and metastasis, both at local sites and premetastatic niches where invasion occurs in distal organs. Taken together, our findings suggest that efforts to target TLR4 on tumor cells may simultaneously quell local and systemic inflammatory pathways that promote malignant progression, with implications for how to prevent tumor recurrence and the establishment of metastatic lesions, either during chemotherapy or after it is completed. Cancer Res; 74(19); 5421–34. ©2014 AACR.

Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Our previous studies have shown that AKR1B10 is secreted through a lysosome-mediated nonclassical pathway, leading to an increase in the serum of breast cancer patients. This study illuminates the regulatory mechanism of AKR1B10 secretion. The cytosolic AKR1B10 associates with and is translocated to lysosomes by heat shock protein 90α (HSP90α), a chaperone molecule. Ectopic expression of HSP90α significantly increased the secretion of endogenous AKR1B10 and exogenous GFP-AKR1B10 fusion protein when cotransfected. Geldanamycin, a HSP90α inhibitor, dissociated AKR1B10-HSP90α complexes and significantly reduced AKR1B10 secretion in a dose-dependent manner. We characterized the functional domain in AKR1B10 and found that helix 10 (amino acids 233-240), located at the C terminus, regulates AKR1B10 secretion. Targeted point mutations recognized that amino acids Lys-233, Glu-236, and Lys-240 in helix 10 mediate the interaction of AKR1B10 with HSP90α. Together, our data suggest that HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. This finding is significant in exploiting the use of AKR1B10 in cancer clinics.

Within the last two decades, the advancement in cellular and cancer biology research can be defined by a few foundational pillars-one of the most important ones being the advent of small-molecule inhibitors (SMIs). Due to their several advantages over contemporary cytotoxic drugs, these engineered molecules have not only shed light on a much deeper understanding of the cellular microenvironment and signal transduction pathways, they have also provided hope of achieving clinical benefits of better patient tolerability while surpassing the efficacy of chemotherapeutic agents. It is, therefore, no surprise that research and development of SMIs has witness a steady surge amongst pharmaceutical industries and research institutions. It is, however, safe to assume that the current applications of SMIs are the mere tip of a yet-inconceivable iceberg. J. Cell. Biochem. 118: 959-961, 2017. © 2016 Wiley Periodicals, Inc.

Female germ cell and its intricate milieu regulate key processes of folliculogenesis and early embryonic development. However, the composition and dynamics of the oocyte transcriptome defines its future fertilizing ability which in turn depends on a number of oocyte specific genes whose identities are still unknown. In this context, the construction of buffalo oocyte specific subtracted cDNA library has raised fresh challenges of defining the importance of a battery of oocyte expressed transcripts in oocyte maturation. The present study tried to characterize these hitherto unknown transcripts and further to assess their expression dynamics in buffalo oocytes of different quality. For this purpose, three ESTs were selected from the library and subjected to 5' and 3' RACE for generating their full length sequences. These constructed full length sequences were validated by amplifying them in oocytes. Further these sequences were extensively analyzed for their coding potential and possible role using coding potential calculator and miRNA database. Besides, their expression was monitored during in vitro maturation in good (BCB+) and poor quality (BCB-) oocytes which was interestingly found to be differing significantly. All the three sequences under study were interpreted as long intergenic non-coding RNAs with the possibility of two of them acting as a miRNA precursors. Also, their differential expression trends in competitively diverse oocytes hints at their possible involvement in oocyte maturation and future embryonic development which needs to be explored further. J. Cell. Biochem. 118: 1712-1721, 2017. © 2016 Wiley Periodicals, Inc.

Acetyl-CoA carboxylases (ACCs) play a rate-limiting role in fatty acid biosynthesis in plants, microbes, mammals and humans. ACCs have the activity of both biotin carboxylase (BC) and carboxyltransferase (CT), catalyzing carboxylation of Acetyl-CoA to malonyl-CoA. In the past years, ACCs have been used as targets for herbicides in agriculture and for drug discovery and development of human diseases, such as microbial infections, diabetes, obesity and cancer. A great number of small molecule ACC inhibitors have been developed, including natural and non-natural (artificial) products. These chemicals target BC reaction, CT reaction or ACC phosphorylation. This article provides a comprehensive review and updates of ACC inhibitors, with a focus on their therapeutic application in metabolic syndromes and malignant diseases. The patent status of common ACC inhibitors is discussed.

Acetyl-CoA carboxylases (ACC) are rate-limiting enzymes in de novo fatty acid synthesis, catalyzing ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA. Malonyl-CoA is a critical bi-functional molecule, i.e., a substrate of fatty acid synthase (FAS) for acyl chain elongation (fatty acid synthesis) and an inhibitor of carnitine palmitoyltransferase I (CPT-I) for fatty acid beta-oxidation. Two ACC isoforms have been identified in mammals, i.e. ACC-alpha (ACCA, also termed ACC1) and ACC-beta (ACCB, also designated ACC2). ACC has long been used as a target for the management of metabolic diseases, such as obesity and metabolic syndrome, and various inhibitors have been developed in clinical trials. Recently, ACCA up-regulation has been recognized in multiple human cancers, promoting lipogenesis to meet the need of cancer cells for rapid growth and proliferation. Therefore, ACCA might be effective as a potent target for cancer intervention, and the inhibitors developed for the treatment of metabolic diseases would be potential therapeutic agents for cancer therapy. This review summarizes our recent findings and updates the current understanding of the ACCA with focus on cancer research.

Inflammatory cells and signals contribute to the initiation and development of cancer. In fact, persistent inflammatory conditions resulting from infection or injury can exist before a normal cell is transformed into a cancer cell. Situations of chronic inflammation can promote genomic instability leading to DNA damage, oncogene activation, or impaired function of a tumor suppressor. Alternatively, cancer development unrelated to inflammation can stimulate the development of an inflammatory microenvironment that promotes tumor cell proliferation. Whether chronic or tumor-derived, inflammation and inflammation-related stimuli within the tumor microenvironment permits proliferation and survival of cancer cells, promotes blood and lymphatic vessel formation, and aids in invasion and metastasis. The inflammatory status of the tumor microenvironment can act to quell the body's natural immune response and effectively ameliorate a positive response to many commonly used anti-cancer antibodies and chemotherapeutic agents. New evidence suggests that the molecular pathways and consequences of inflammation specifically related to the tumor microenvironment are starting to be understood. This new information implicates novel cellular targets that could lead to improved diagnosis and treatment for a variety of solid malignancies.

We analyze the deformation of the substrate matrix and the shear forces it is exposed to through the geometry of graphite flakes in the substrate from samples already subjected to breaking pressure applications up to 30 bar. Microscopic Images of the cross-section of the brake pad polished to a 0.1 μm scale were examined. Statistical analysis was conducted, state vectors and variance-co-variance matrix were defined, and considered every state as a distribution. Parameters for each “shear deformation stage” were estimated using Expectation-Maximization algorithm. Finally, a mixture model was identified for the distribution of graphite flakes across the rotor thickness.Copyright © 2015 by ASME

The effect of an applied electric potential on the dynamics of gas bubble formation from a single nozzle in glycerol was studied experimentally. Dry nitrogen was bubbled into glycerol through a nozzle having an electrified tip while pressure measurements were made upstream of the nozzle. As the applied electric potential was increased from zero, bubble size reduced, bubble shape became more spherical, and bubbling frequency increased. At constant gas flow, bubble-formation exhibited a classic period-doubling route to chaos with increasing potential. We defined an electric Bond number assuming that both the liquid and gas phases are conducting. This is in contrast to previous studies where one phase was considered a perfect conductor and the other one a perfect nonconductor or insulator. Although electric potential and gas flow appear to have similar effects on the period-doubling bifurcation process for this system, the relative impact of electrostatic forces, as measured in terms of electric Bond number for conducting liquid and gas phases, is smaller. However, the relative impact of electrostatic forces for the case of insulating liquid and conducting gas phases is comparable to flow forces. Further data collection is required for different nozzle geometries and liquid column heights in order to verify the relative impacts of electrostatic and flow forces, and would allow us to ascertain if electrostatic potential is a feasible manipulated variable for controlling this system.

Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC. Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules. Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6. Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway. Citation Format: Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2017-1078

Titanium alloy (Ti6Al4V (ASTM F-136)) is typically used for modular hip implant stems. This highly corrosion resistant alloy forms passive surface oxide films spontaneously. However, with modular designs, micro-motion may occur at the taper junctions during mechanical loading. Complex physical/chemical reactions take place which may result in pitting and crevice corrosion. Crevices between the taper junctions may allow the body fluids to enter and remain stagnant. These conditions make the modular tapers susceptible to fatigue and mechanically assisted crevice corrosion. When two or more surfaces are in close proximity, it leads to the creation of a locally blocked region in which enhanced dissolution may occur. The in vivo degradation of metal alloy implants compromises the structural integrity. Stochastic modeling of crevice corrosion is performed based on the mechanism behind the phenomenon. Sensitivity analysis is performed, and conclusions drawn.

In this study genome-wide di-methylated H3K4 (H3K4me2) and tri-methylated H3K27 (H3K27me3) modification profiles were analyzed in spermatozoa of buffalo bulls having wide fertility differences. The custom designed 4 × 180 K buffalo (Bubalus bubalis) ChIP-on-chip array was fabricated by employing array-based sequential hybridization using bovine and buffalo genomic DNA for comparative hybridization. The buffalo specific array developed had 177,440 features assembled from Coding sequences, Promoter and CpG regions comprising 2967 unique genes. A total of 84 genes for H3K4me2 and 80 genes for H3K27me3 were found differentially enriched in mature sperm of high and sub-fertile buffalo bulls. Gene Ontology analysis of these genes revealed their association with different cellular functions and biological processes. Genes identified as differentially enriched between high and sub-fertile bulls were found to be involved in the processes of germ cell development, spermatogenesis and embryonic development. This study presents the first genome-wide H3K4me2 and H3K27me3 profiling of buffalo bull sperm. Results provide a list of specific genes which could be made responsible for differential bull fertility.

Associations between abnormal methylation of spermatozoan DNA with male infertility have been sought in recent years to identify a molecular explanation of differential spermatozoan function. The present work was undertaken to investigate the methylation profile of differentially methylated regions (DMRs) in the IGF2-H19 locus of Bos taurus X Bos indicus crossbred bull spermatozoa. Bulls having more than at least 100 insemination records over a period of 12 years were classified into two groups of five bulls each belonging to low- and high-fertility groups. The IGF2 and H19 DMR sequences in B. indicus cattle were observed to be in absolute homology with B. taurus cattle. The DNA of crossbred bull spermatozoa was isolated, bisulfite treated, and amplified for specific DMR regions using methylation-change-specific primers. The overall degree of methylation at IGF2-H19 DMRs was not found to be significantly different among two groups of bulls. The sixth CTCF binding site (CCCTC) identified in H19 DMR, however, had a significant methylation difference between the high- and low-fertility bulls. It was concluded that alteration of the methylation levels at IGF2-H19 DMRs might not be responsible for the fertility difference of crossbred bulls, although the role played by the specific CTCF binding sites at this locus, which could influence IGF2 expression during spermatogenesis and early embryonic development, deserves further attention.

Overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. We found that TLR4 is overexpressed in the majority of clinical breast cancer samples and in 68% of the examined breast cancer lines. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug paclitaxel. LPS is frequently used to show a tumor-promoting role of TLR4 although this bacterial component is unlikely to be found in the breast cancer environment. We reasoned that paclitaxel-dependent activation of TLR4 is more relevant to breast cancer chemoresistance that could be mediated by activation of the NF-κB pathway leading to upregulation of prosurvival genes. To test this hypothesis, we correlated TLR4 expression with resistance to paclitaxel in two modified breast cancer lines with either depleted or overexpressed TLR4 protein. Depletion of TLR4 in naturally overexpressing MDA-MB-231 cells downregulated prosurvival genes concomitant with 2- to 3-fold reduced IC50 to paclitaxel in vitro and a 6-fold decrease in recurrence rate in vivo. Conversely, TLR4 overexpression in a negative cell line HCC1806 significantly increased expression of inflammatory and prosurvival genes along with a 3-fold increase of IC50 to paclitaxel in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. Importantly, both tumor models showed that many paclitaxel-upregulated inflammatory cytokines were coinduced with their receptors suggesting that this therapy induces autocrine tumor-promoting loops. Collectively, these results show that paclitaxel not only kills tumor cells but also enhances their survival by activating TLR4 pathway. These findings suggest that blocking TLR4 could significantly improve response to paclitaxel therapy. Mol Cancer Ther; 12(8); 1676–87. ©2013 AACR.

Background: Limitless replicative potential is one of the hallmarks of cancer which occur due to dysregulated cell cycle. Cyclin dependent kinases (CDKs) are major regulators of mammalian cell cycle, which are often hyperactivated in cancer. Therefore, inhibition of cyclin dependent kinases represents an attractive therapeutic approach in cancer therapy. Dinaciclib is a CDK1, CDK2, CDK5 and CDK9 inhibitor, currently being tested in clinical trials in patients with advanced malignancies. The goal of this project was to determine the efficacy and the mechanisms of action of Dinaciclib in triple negative breast cancer (TNBC) by using patient derived xenografts (PDXs).The study results will provide more insights in identification of predictive biomarkers that are important for its clinical development. Methods: The efficacy of Dinaciclib was determined in 3 TNBC PDXs and 3 established TNBC cell lines by in vitro cytotoxic assays and calculating the IC50. Colony formation assay was also performed in 3 dimensional matrigel matrixes to confirm in vitro cytotoxic assay results. Cell cycle analysis was done using PI staining to determine the percentage of cells in G2/M phase after Dinaciclib treatment. Apoptosis analysis was done by Annexin V and PI double staining using FACS. Effect of CDK1, CDK2, and CDK9 on cell proliferation and Dinaciclib efficacy was determined by knocking down these proteins using CRISPER-CAS plasmid. Efficacy of Dinaciclib on tumor growth inhibition was also determined in vivo by implanting PDX cell lines in nude mice. Results: All the PDXs and established breast cancer cell lines tested were sensitive to Dinaciclib as demonstrated by IC50 value ranging from 1 to 20nM. Dinaciclib effectively inhibited the colony formation in 3D culture in all the PDXs and established breast cancer cell lines at 10nM dose. Dinaciclib significantly caused cell cycle arrest at G2/M phase after 24 hours of treatment. It also caused apoptosis induction after 24 hours of treatment. Dinaciclib reduced the CyclinB1, phospho-CDK1, Survivin and MYC protein levels after 24 hour of treatment as analyzed by western blotting. Knockdown of CDK1 and CDK2 inhibited cell proliferation by 30% and 50% in MDA-MB-231 cells in vitro. Dinaciclib significantly inhibited PDX tumor growth in vivo. Conclusions: These data suggest that Dinaciclib is effective in inhibiting triple negative breast cancer growth in vitro and in vivo. Dinaciclib induced both cell cycle arrests at G2/M phase and apoptosis in breast cancer cells. Dinaciclib targets multiple proteins like CDK1, CDK2, CyclinB1 and cMYC, which can be further validated as predictive biomarkers. Dinaciclib can be used as a potential monotherapy for TNBC patients in clinics. Citation Format: Sandeep Rajput, Zhanfang Guo, Cynthia Ma. CDKs inhibitor: potential monotherapy for treatment of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3097. doi:10.1158/1538-7445.AM2015-3097

A Principal Curve is a hypercurve that passes through the center of the data cloud. We adapt and expand the principal curve algorithm to develop a non‐parametric approach called Cluster‐linked Principal Curves (CLPC) that locally approximates the structure and scatter in a distribution of data points. The iterative algorithm is based on Expectation‐Maximization (E‐M) principle. The projections of data points on the principal curve or arc lengths are capable of characterizing the data in fewer dimensions and with greater accuracy than PCA. The distribution of arc lengths is used for gauging stationarity and reversibility, and monitoring. For illustration we use the embeddings formed from chaotic gas pressure time series measurements collected before an electrified capillary nozzle that injects bubbles into a liquid‐filled column.

Background: Lymph node (LN) metastasis, the strongest prognostic factor for breast cancer (BC) patient survival, directly correlates with increased density of lymphatic vessels. The formation of new lymphatic vessels (i.e. lymphangiogenesis) is strongly promoted by tumor-associated macrophages (TAMs) through mechanisms that are not fully understood. We recently discovered that inflammatory lymphangiogenesis is driven by bone marrow derived myeloid cells that transdifferentiate into lymphatic-like cells and structurally contribute to growing lymphatic vasculature. Clinical BC are characterized by an inflammatory environment and massive recruitment of macrophages. We, therefore, hypothesize that BC mobilized monocytic precursors may promote tumor lymphangiogenesis by induction of their transdifferentiation into Macrophage-derived Lymphatic Endothelial Cell Progenitors designated here as M-LECPs. Methodology: To test this hypothesis, we analyzed 75 clinical specimens of invasive breast carcinoma and 5 normal breast tissues for the presence of M-LECPs and incidence of their integration into lymphatic vessels. M-LECPs were identified by double or triple staining using multiple specific markers for myeloid (CD68, CD14, and CD11b) and lymphatic lineages (LYVE-1, podoplanin and VEGFR-3). Integrated M-LECPs were identified by double staining of LYVE-1+ vascular structures with antibodies against myeloid-specific markers as well as confocal microscopy using anti-VE-cadherin staining. Results: Out of 75 tumors, 58% had lymphatics, and 46% of these specimens showed integration of M-LECPs in the lymphatic vessels. In stark contrast, normal mammary tissues showed less than 5% of either double-stained macrophages or dual-identity vessels. The predominant M-LECP association with tumors was also evident in metastatic BC mouse models including MDA-MB-231 (human xenograft), R3L (transplantable syngeneic), and MMTV-PyMT (spontaneous syngeneic). All three models contained 40-50% of TAMs positive for lymphatic markers and up to 90% of lymphatic vessels positive for myeloid markers. In vitro studies showed that inflammatory triggers induce lymphatic proteins in myeloid cells but not myeloid markers in inflamed lymphatic endothelial cells. Conclusions/Significance: These findings show, for the first time, that majority of TAMs in clinical BC exhibit the lymphatic phenotype and integrate into lymphatic vessels. Reproduction of this phenomenon in three BC mouse models and cultured myeloid cells treated with inflammatory mediators suggests a strong self-autonomous role of TAMs into outgrowth of tumor lymphatics. Further investigation of this novel mechanism of TAM-dependent lymphangiogenesis may identify new targets for inhibiting lymphatic metastasis leading to improved survival of breast cancer patients. Citation Format: Kelly Hall, Lisa Volk-Draper, Sandeep Rajput, David DeNardo, Sophia Ran. Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 12. doi:10.1158/1538-7445.AM2014-12

Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2014-771

Introduction: The basaloid triple-negative breast cancer (B-TNBC) is one of the most aggressive, therapy-resistant, and metastatic tumors. Current models do not recapitulate the basaloid phenotype of TNBC, thus limiting the understanding of its biology and designing new treatments. We identified HCC1806 as a line expressing typical B-TNBC markers, engineered a subline with traceable reporters, and determined growth, drug sensitivity, recurrence, and vascular and metastatic patterns of orthotopic xenografts in immunodeficient mice. Methods: mRNA and protein analyses showed that HCC1806 expresses basal but not luminal or mesenchymal markers. HCC1806-RR subline stably expressing red fluorescent protein and Renilla luciferase was generated and characterized for sensitivity to chemodrugs, orthotopic growth, vascular properties, recurrence, metastasis, and responsiveness in vivo . Results: The HCC1806 cells were highly sensitive to paclitaxel, but cytotoxicity was accompanied by pro-survival vascular endothelial growth factor-A loop. In vivo , HCC1806-RR tumors display linear growth, induce peritumoral lymphatics, and spontaneously metastasize to lymph nodes (LNs) and lungs. Similarly to human B-TNBC, HCC1806-RR tumors were initially sensitive to taxane therapy but subsequently recur. Bevacizumab significantly suppressed recurrence by 50% and reduced the incidence of LN and pulmonary metastases by, respectively, 50% and 87%. Conclusions The HCC1806-RR is a new model that expresses bona fide markers of B-TNBC and traceable markers for quantifying metastases. Combination of bevacizumab with nab-paclitaxel significantly improved the outcome, suggesting that this approach can apply to human patients with B-TNBC. This model can be used for defining the metastatic mechanisms of B-TNBC and testing new therapies.

Background: Paclitaxel elicits both cytotoxic and pro-survival responses in tumor cells. The tumor-promoting effect of paclitaxel is a currently unrecognized determinant for decreasing the apoptotic effect of paclitaxel therapy. The likely mechanism for paclitaxel-dependent tumor-activating effects is the ability of paclitaxel to activate Toll-like Receptor-4 (TLR4) pathway. TLR4 is often over-expressed in malignant epithelial cells in which its signaling hyper-activates NF-kB, MAPK and PI3K pathways providing a pro-survival benefit to the residual cancer cells. The goal of this project was to identify the TLR4 role in paclitaxel resistance by correlating the expression profile of TLR4 pathway in a panel of breast carcinoma cells with sensitivity to paclitaxel therapy. Methods: TLR4 mRNA levels were determined in 18 breast carcinoma lines by qRT-PCR. The functionality of TLR4 pathway was determined by measuring mRNA of downstream products (IL-6, IL-8, MCP-1 and TNFα) after stimulating cells with a natural TLR4 ligand, lipopolysaccharide (LPS), or paclitaxel. Protein levels were measured by ELISA. Cytotoxic assays were used to correlate the level of TLR4 expression and responsiveness to paclitaxel. TLR4-positive (MDA-MB-231) and negative (HCC1806) lines were engineered to stably down-regulate and over-express TLR4, respectively. The target expression in modified clones was determined by qRT-PCR and Western Blot. The modified lines were analyzed for functionality and altered responsiveness to paclitaxel using the aforementioned methods. Results: TLR4 was expressed in 60% of human breast cancer cell lines. TLR4 receptor in MDA-MB-231 line was functional as demonstrated by up-regulation of inflammatory cytokines. LPS increased IL-6 and MCP-1 by 8-10 fold whereas IL-8 and TNF-alpha were increased by 120-300 fold. Paclitaxel had 1/10 to 1/3 of the LPS activity. MDA-MB-231 line was ∼5 fold more resistant to paclitaxel than HCC1806 lacking TLR4. Paclitaxel treatment not only drastically increases secretion of NF-kB dependent cytokines but also up-regulated the expression levels of their receptors suggesting establishment of novel autocrine pro-survival and proliferative positive loops. Anti-TLR4 antibody inhibited paclitaxel stimulating effects by nearly 100%. Conclusions: These data show that paclitaxel up-regulates both inflammatory cytokines and their receptors in human breast carcinoma cells, likely through activation of the TLR4 pathway. Inflammatory pathway signaling increases survival and proliferation in TLR4-positive cells, suggesting that activation of this pathway in malignant cells maintain chronic inflammation and promote tumor growth and metastasis through both paracrine and autocrine loops. This study suggests that tumor resistance to paclitaxel might be determined by TLR4 expression, and that blocking TLR4 might significantly improve tumor response to paclitaxel therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1700. doi:10.1158/1538-7445.AM2011-1700

Background: Toll like receptor 4 (TLR4) is a transmembrane receptor expressed in macrophages that plays a major role in pathogen recognition and activation of innate immunity. TLR4 signaling has been linked to tumorigenesis and promotion of metastasis in ovarian, lung, prostate and head-neck cancers through up-regulation of the expression of inflammatory cytokines (e.g., IL-6, IL-8 and TNF-alpha). These factors increase cancer cell survival and resistance to chemotherapy through up-regulation of anti-apoptotic and down-regulation of apoptotic proteins, respectively. The goal of this study was to generate new isogenic cell lines that would allow analysis of the TLR4 role in paclitaxel resistance in breast cancer. To this end, we used TLR4-positive and negative lines (MDA-MB-231 and HCC1806, respectively) to generate stable TLR4 knockdown and over-expressing line derivatives. Methods: To generate a TLR4 knockdown line, MDA-MB-231 cells were transfected with pisiRNA-TLR4 plasmid and selected with zeocin (50μg/ml). To generate a TLR4 over-expressing line, HCC1806 cells were transfected with pKT2-TLR4-PURO plasmid and selected with puromycin (1μg/ml). Ten to 20 monoclonal colonies were selected from each line and characterized for TLR4 expression and TLR4-dependent cell activities by qRT-PCR and western blot. Results: TLR4 mRNA expression was reduced by >90% as compared with parental line in five out of 8 MDA-MB-231TLR4− clones examined. One clone had 60% reduction and two clones had no effect on TLR4 mRNA determined by qRT-PCR. TLR4 mRNA was up-regulated 1,000-12,000 fold in five (out of 11) clones derived from HCC1806 line. Two clones overexpressed TLR4 mRNA by 300-500 fold and four clones had increased TLR4 expression by 1-6 fold. All selected clones were analyzed for TLR4 functional signaling by exposure to LPS (1μg/ml), which was reduced by 50-70% in MDA-MB-231TLR4− clones and increased by 15-25 folds in HCC1806TLR4+ sub-lines, as compared with cells transfected with empty vectors. Conclusions: These data show that we successfully created sub-lines of MDA-MB-231 with stable knockdown of TLR4 and sub-lines of breast carcinoma HCC1806 line with stable over-expression of TLR4. Ectopically expressed human TLR4 as well as TLR4 downregulating sequence had significant functional effects on the TLR4 signaling as demonstrated by increase and decrease of LPS-activated targets (IL-6, IL-8 and TNF-alpha) in MDA-MB-231TLR4− and HCC1806TLR4+ sub-lines, respectively. These new sub-lines will serve as excellent tools for analyzing the role of the TLR4 pathway signaling in breast cancer progression, metastasis and resistance to therapy. * These authors contributed equally in this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5585. doi:10.1158/1538-7445.AM2011-5585