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The development of network inference methodologies that accurately predict connectivity in dysregulated pathways may enable the rational selection of patient therapies. Accurately inferring an intracellular network from data remains a very challenging problem in molecular systems biology. Living cells integrate extremely robust circuits that exhibit significant heterogeneity, but still respond to external stimuli in predictable ways. This phenomenon allows us to introduce a network inference methodology that integrates measurements of protein activation from perturbation experiments. The methodology relies on logic-based networks to provide a predictive approximation of the transfer of signals in a network. The approach presented was validated in silico with a set of test networks and applied to investigate the epidermal growth factor receptor signaling of a breast epithelial cell line, MFC10A. In our analysis, we predict the potential signaling circuitry most likely responsible for the experimental readouts of several proteins in the mitogen activated protein kinase and phosphatidylinositol-3 kinase pathways. The approach can also be used to identify additional necessary perturbation experiments to distinguish between a set of possible candidate networks.

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Objective: To investigate the effects of hyperbaric oxygen (HBO 2 ) on aggressive periodontitis (AgP), and subgingival obligate anaerobes in Chinese patients. Materials and Methods: Sixty cases of Chinese patients with AgP were randomly divided into two groups -the HBO 2 group (30 cases) and the control group (30 cases). Study teeth were divided into four groups -: the HBO 2 therapy, the HBO 2 + scaling scaling group, the scaling group and the control group. Subgingival anaerobic organisms were measured with anaerobic culture, and number of obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus was counted. Comparisons of changes in the clinical indices, and subgingival anaerobes were made between the groups. Results: Highly significant differences in gingival index (GI), probing depth (PD), attachment loss (AL), and Plaque index (PLI), and tooth odontoseisis (TO) were seen in the HBO 2 , the HBO 2 + scaling and the scaling groups when compared with the control group (P

High-throughput phenotypic screens that incorporate compound biochemical activity annotations are positioned for novel target discovery in cancer. We used machine learning approaches to correlate kinome-wide profiling data of a collection of kinase inhibitors with phenotypic cell proliferation data for the same compounds. We assembled a library of profiled kinase inhibitors with diverse chemotypes and kinome-wide selectivities and determined their anti-proliferative activities in a panel of sarcoma cell lines. Using a previously published machine learning algorithm, we related the compound inhibition profiles across 237 kinases to their abilities to inhibit cell proliferation. This identified Protein Kinase D (PRKD) as a putative novel target kinase selectively in synovial sarcoma cell lines, such that its inhibition leads to a decrease in proliferation in these cells. Next, we reasoned that our approach could be leveraged to identify targets that, when co-inhibited, induce a synergistic phenotype. This would enable rational design of drug combinations for further testing as opposed to the labor-intensive, random, pairwise testing commonly performed. To identify targets synergistic with PRKD, we performed a screen of a synovial sarcoma cell line in the presence of a selective PRKD inhibitor. Several kinases became prioritized as new targets in this “synergy screen.” Combining selective inhibitors for each synergistic target, as defined by Chou-Talalay, (here, CDK and AKT) with PRKD inhibitors synergistically reduced synovial sarcoma cell proliferation. Conversely, combining PRKD inhibitors with a selective inhibitor of a kinase that was deprioritized in the synergy screen (p38 MAPK) resulted in non-synergistic or even antagonistic effects. Overall, our approach provides a promising framework to identify new targets of cancer subtypes and a novel methodology to identify new combinational strategies for treatment. Citation Format: Eric J. Lachacz, Zhi Fen Wu, John L. Bixby, Vance P. Lemmon, Sofia D. Merajver, Hassan Al-Ali, Matthew B. Soellner. Identifying drug targets in sarcoma using machine learning and cell phenotype-based compound screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4647.

One of the major clinical challenges in breast cancer treatment is the rapid development of resistance to targeted therapies. In light of this, there is significant interest in identifying markers of drug resistance. In particular, markers of drug resistance are sought that can be targeted to overcome acquired drug resistance. To this end, we have used reverse phase protein array (RPPA) analysis to identify differences in protein expression levels between cell lines that are sensitive or have acquired resistance to UM-164, a mechanistically novel, dual c-Src/p38 kinase inhibitor with potent anti-TNBC activity. UM-164 resistant cells acquire increased resistance also to many diverse chemotherapeutics and targeted agents relative to their drug-sensitive counterparts. We analyzed RPPA data for both UM0164-sensitive and UM-164-resistant cells and identified an increase in phospho-AKT, phospho-ERK, and phospho-FAK levels in the resistant cells. Using inhibitors of each upregulated pathway, we found that combination of UM-164 with MK2206, an inhibitor of AKT, resulted in an increased sensitization of cells to treatment with UM-164. Furthermore, formal Chou-Talalay synergy analyses demonstrate that AKT inhibitors + UM-164 are effectively synergistic in reducing the proliferation of drug-resistant cells. We further evaluated this combination treatment in mice, combining UM-164 and MK2206 in a xenograft of UM-164-resistant cells, showing significant anti-tumor efficacy. Finally, we show that the combination treatment resulted in increased cell death in ex vivo cultures of patient derived TNBC materials. Together, we have demonstrated that hyperactivation of AKT leads to UM-164 resistance and AKT inhibitors can be used to re-sensitize TNBC cells to UM-164. These results demonstrate that RPPA analyses are a valuable tool for the systematic and unbiased identification of truly actionable markers of therapeutic resistance. Citation Format: Nathan M. Merrill, Eric J. Lachacz, Rabia A. Gilani, LiWei Bao, Zhi Fen Wu, Sofia D. Merajver, Matthew B. Soellner. AKT inhibition sensitizes TNBC cells with acquired drug resistance to UM-164 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 915.

Purpose. Pomegranate fruit extracts (PFEs) possess polyphenolic and other compounds with antiproliferative, pro-apoptotic and anti-inflammatory effects in prostate, lung, and other cancers. Because nuclear transcription factor-kB (NF-kB) is known to regulate cell survival, proliferation, tumorigenesis, and inflammation, it was postulated that PFEs may exert anticancer effects at least in part by modulating NF-kB activity. Experimental design. The authors investigated the effect of a novel, defined PFE consisting of both fermented juice and seed oil on the NF-kB pathway, which is constitutively active in aggressive breast cancer cell lines. The effects of the PFE on NF-kB—regulated cellular processes such as cell survival, proliferation, and invasion were also examined. Results. Analytical characterization of the bioactive components of the PFE revealed active constituents, mainly ellagitannins and phenolic acids in the aqueous PFE and conjugated octadecatrienoic acids in the lipid PFE derived from seeds.The aqueous PFE dose-dependently inhibited NF-kB—dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while decreasing RhoC and RhoA protein expression. Conclusion. Inhibition of motility and invasion by PFEs, coincident with suppressed RhoC and RhoA protein expression, suggests a role for these defined extracts in lowering the metastatic potential of aggressive breast cancer species.

The cellular and molecular events of periodontal healing are coordinated and regulated by an elaborate system of signaling molecules, pointing to a primary strategy for functional periodontal compartment regeneration to replicate components of the natural cellular microenvironment by providing an artificial extracellular matrix (ECM) and by delivering growth factors. However, even with optimal carriers, the localized delivery of growth factors often requires a large amount of protein to stimulate significant effects in vivo, which increases the risk and unwanted side effects. A simple and relatively new approach to bypassing this dilemma involves converting cells into protein producing factories. This is done by a so-called gene delivery method, where therapeutic agents to be delivered are DNA plasmids that include the gene encoding desired growth factors instead of recombinant proteins. As localized depots of genes, novel gene delivery systems have the potential to release their cargo in a sustained and controlled manner and finally provide time- and space- dependent levels of encoded proteins during all stages of tissue regrowth, offering great versatility in their application and prompting new tissue engineering strategy in periodontal regenerative medicine. However, gene therapy in Periodontology is clearly in its infancy. Significant efforts still need to be made in developing safe and effective delivery platforms and clarifying how gene delivery, in combination with tissue engineering, may mimic the critical aspects of natural biological processes occurring in periodontal development and repair. The aim of this review is to trace an outline of the state-of-the-art in the application of gene delivery and tissue engineering strategies for periodontal healing and regeneration.

Background: Aggressive periodontitis (AgP) has a genetic basis. It has been reported that the functional gene polymorphisms of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-2 (TIMP-2) alter their expressions in transcriptional level and they are involved in the tissue destruction of periodontitis. The study was carried out to analyse the association of functional polymorphisms in MMP-2, MMP-9 and TIMP-2 with generalized AgP (G-AgP) in a Chinese population. Material and Methods: The study population consisted of 79 Chinese patients with G-AgP and 128 healthy controls. DNA was obtained from oral mucosa swab samples. MMP-2 genotypes were determined by PCR-based denaturing high-performance liquid chromatography analysis while MMP-9 and TIMP-2 genotypes were identified by a PCR-based restriction fragment length polymorphism. χ2 test after Yates' correction was used to investigate the possible association of the genotypes with the G-AgP. Results: Although gene polymorphisms for MMP-2 and MMP-9 did not show any association with the G-AgP, the analysis of the TIMP-2 −418G to C gene polymorphism revealed significant differences between the patients and controls. Compared with controls, a significant increasing trend of TIMP-2 −418C carrier in the G-AgP patients occurred (p=0.013). Conclusion: It is suggested that the TIMP2 −418G to C gene polymorphism is associated with G-AgP in the Chinese subjects.

Novel thermomechanical hydrogel scaffolds containing our previously prepared microspheres loaded with bone morphogenetic proteins (BMP) were successfully generated by radical crosslinking and low dose γ-irradiation from combination of two kind of biomaterials: glycidyl methacrylated dextran (Dex-GMA) and gelatin. The structure of those resulting smart hybrid hydrogels was evaluated by mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM) analyses, and as a function of the degree of Dex-GMA's substitution (DS), the proportion between Dex-GMA and gelatin, and the initial polyethyleneglycol (PEG) concentration used in the preparation of the hydrogels. The swelling and degradation properties and the temperature-sensitive drug release manner were determined by dynamic evaluation methods in vitro , and the gel content was also calculated. MIP analysis showed that by systematically altering the preparation parameters, the overall networks were clearly macroporous with pore sizes ranging from 5.6 ± 4.2 to 37.7 ± 13.7 μm. As expected, the pore size decreased as DS and initial PEG concentration increased, whereas the opposite was found for the gel content. Moreover, the porosity values ranged from 73.7 ± 12.4% up to 89.6 ±6.3%. The SEM results also showed the inter-connective pores as well as microspheres encased into their porous structure of those hydrogels. The swelling and degradation properties of the resultant hydrogels varied according to the DS of Dex-GMA and initial PEG concentration, while the proportion between Dex-GMA and gelatin had no significant influence on those characterizations. By changing the composition ratio of the two precursors, the phase transition temperature (lower critical solution temperature, LSCT) of the hydrogel scaffolds could also be adjusted to be or near the body temperature, so BMP release from microsphere–hydrogel compounds could be accordingly controlled and the release period could be varied from 18 to more than 28 days. These results demonstrated that a novel temperature-sensitive and biodegradable Dex-GMA/gelatin scaffold containing microspheres loaded with BMP could be successfully developed from both dextran- and gelatin-based biomaterials, which could promisingly satisfy the need, desire, and expectation of both self-regulated drug delivery and tissue-engineering applications.

Dynamic interactions and dissolution of cell-extracellular matrix contacts are required steps to support cell growth and survival during cancer cell metastasis. Malignant cells acquire the ability to remodel extracellular matrix (ECM) and to modulate the expression of ECM receptors. Integrins are cellular receptors for molecules in the extracellular matrix. Integrin signaling is known to regulate metastatic cancer phenotypes by interacting synergistically with several signaling pathways, including the growth factor receptor pathway, the Ras-MAP kinase (Ras-MAPK) pathway and the Rho-effector pathway. In this mini-review, we discuss the functions of the Rho proteins and their relationship with other signaling pathways in matrix remodeling and integrin signaling of highly motile and invasive cancer cells.

The present work focused on the design of novel hydrogel microspheres based on both dextran- and gelatin-derived biomaterials, and discussed whether locally controlled delivery of IGF-I from dextran-co-gelatin hydrogel microspheres (DG-MP) was useful for periodontal regeneration enhancement. Microspheres were synthesized when gelatin was cooperating with glycidyl methacrylate (GMA) derivatized dextrans (Dex-GMA) and the resultant DG-MP with a hydrogel character of which the cross-linking density could be controlled by the degree of substitution (DS, the number of methacrylates per 100 glucopyranose residues) of Dex-GMA. In this study, three types of DG-MP (DG-MP4.7, DG-MP6.3 and DG-MP7.8) obtained from gelatin and Dex-GMA (differing in DS: 4.7, 6.3 and 7.8 respectively) were prepared and characterized by swelling and degradation properties, drug release kinetics and biological capability in promoting tissue regeneration. By swelling in aqueous positively charged IGF-I solutions, the protein could be encapsulated in DG-MP by polyionic complexation with negatively charged acidic gelatin. No obvious influence of Dex-GMA's DS on DG-MP's configuration and size was observed, and the release and degraded properties showed no significant difference between three types of DG-MP in PBS buffer either. However, high DS of Dex-GMA could lower microsphere's swelling, prolong its degraded time and minimize IGF-I burst release markedly in dextranase-containing PBS, where IGF-I release from a slow release type of microspheres (DG-MP7.8) could be maintained more than 28 days, and an effective protein release kinetics without a significant burst but a relevantly constant release after the initial burst was achieved. IGF-I in DG-MP resulted in more new bone formation in the periodontal defects within 4 or 8 weeks than IGF-I in blood clot directly did (P < 0.01). The observed newly formation of periodontal tissues including the height and percentage of new bone and new cementum on the denuded root surfaces of the furcation area in DG-MP7.8 group were more than that in other groups (P < 0.05). The adequate width of regenerative periodontal ligament (PDL), regular Sharpey's fibers and alveolar bone reconstruction could be observed only in DG-MP7.8 group. These combined results demonstrate that effective release kinetics can be realized by adjusting the DS of Dex-GMA and followed cross-linking density of DG-MP, and that locally controlled delivery of IGF-I from slow release type of DG-MP may serve as a novel therapeutic strategy for periodontal tissue regeneration.

Recent developments of biotechnology have produced a great variety of protein and bioactive drugs. For these drugs to be used therapeutically, suitable drug delivery systems have become increasingly essential. Dextran-derived biomaterials have been considered to be compatible matrices for protein and bioactive drugs because of their hydrophilic properties and ability to control drug dissolution and permeability. A novel class of dextran-glycidylmethacrylate (Dex-GMA)/poly(ethylene glycol) (PEG) microspheres were designed and synthesized by polymerization of Dex-GMA emulsified in an aqueous PEG solution. Dex-GMA was prepared by substituting the hydroxyl groups in Dex by GMA. The drug loading and in vitro drug release was evaluated by routine procedure and the biological activity of BMP-loaded microspheres was studied by experimental cytology methods. Recombinant human bone morphogenetic protein-2 (rhBMP-2) were entrapped in dextran-derived microspheres quantitatively and with full preservation of their biological activity. In vitro release kinetics indicated that dextran-derived microspheres could retain rhBMP-2 in a variable manner depending on the preparation and degradation of the microspheres. The release profiles of rhBMP-2 from microspheres as a function of time showed that rhBMP-2 releasing kinetics in vitro fitted to first-order and Higuchi equations. The release profile in vitro was in accord with two phases kinetics law and more than 60% drug were released during 20 days. Cytology studies showed rhBMP-2 microspheres have good biological effects on cultured periodontal ligament cells, and could achieve a longer action time than concentration of rhBMP-2 solution. These properties make those microspheres interesting osteo-conductive BMP carriers, allowing to decrease the amount of implanted factor required for tissue regeneration.

Aim: To prepare new pharmaceutical forms with sustained delivery properties of recombinant human bone morphogenetic protein-2 (rhBMP2) for tissue engineering and guided tissue regeneration (GTR) use. Methods: rhBMP2-loaded dextran-based hydrogel microspheres (rhBMP2-MPs), which aimed to keep rhBMP 2 bioactivity and to achieve long-term sustained release of rhBMP 2 , were prepared by double-phase emulsified condensation polymerization. The physical, chemical performances and biological characteristics of those microspheres were studied both in vitro and in vivo . Results: The microspheres' average diameter was 30.33plusminus4.32 mum with 75.4% ranging from 20 mum to 40 mum and the drug loading and encapsulation efficiency were 7.82% and 82.25%, respectively. The rhBMP 2 -releasing profiles in vitro showed that rhBMP 2 release could be maintained more than 10 d. The rhBMP 2 -MPs, with good swelling and biodegradation behavior, could be kept for 6 months at below 4°C without significant characteristic change or bioactivity loss. Cytology studies showed that rhBMP 2 -MPs could promote the proliferation of periodontal ligament cells (PDLCs) approximately 10 d, while the bioactivity of concentrated rhBMP 2 solution could keep no more than 3 d. Scanning electron microscope showed that rhBMP 2 -MPs could be enchased into the porous structure of calcium phosphate ceremic (CPC) and the eugonic growth of PDLCs in CPC/rhBMP 2 -MPs scaffolds. Animal experiments indicated that using CPC/rhBMP 2 -MPs scaffolds could gain more periodontal tissue regeneration than using rhBMP 2 compound firsthand with CPC (CPC/rhBMP 2 ). Conclusion: By encapsulating rhBMP 2 into dextran-based microspheres, a small quantity of rhBMP 2 could achieve equivalent effects to the concentrated rhBMP 2 solution and at the same time, could prolong rhBMP 2 retention both in vitro and in vivo .

Summary Background. There is a need for reliable predictors of breast cancer aggressiveness that will further refine the staging classification and help guide the implementation of novel therapies. We have identified RhoC as being nearly always overexpressed in the most aggressive form of breast cancer, inflammatory breast cancer (IBC); in subsequent work we identified RhoC to be a promising marker of aggressive behavior in breast cancers less than 1 cm in diameter. We hypothesized that RhoC expression would identify aggressive, non-IBC tumors breast cancer patients at any stage with worse outcomes defined as recurrence and/or metastasis. Methods. We constructed four high-density tissue microarrays (TMAs) using 801 tissue cores from 280 patients. These tissues represent a wide range of normal breast and breast disease, including intraductal hyperplasia, ductal carcinoma in situ (DCIS), invasive carcinomas, and distant metastases. The TMAs were immunostained using a polyclonal anti-RhoC antibody developed in our laboratory. Cytoplasmic RhoC expression was scored as negative, weak, moderate, or strong by a previously validated scoring schema. Results. RhoC expression increases with breast cancer progression. All samples of normal breast epithelium had negative to weak staining, whereas staining intensity increased in hyperplasia, DCIS, invasive carcinoma, and metastases (Kruskal–Wallis p < 0.001). In patients with invasive carcinoma, high RhoC expression was associated with features of aggressive behavior including high histologic grade, positive lymph nodes, and negative hormonal receptor status. High RhoC expression was a predictor of overall survival in patients with breast cancer (log rank test, p= 0.002) and was associated with 100% increase in the risk of death as compared to patients with low RhoC expression. Importantly, high RhoC was an independent predictor of poor response to doxorubicin-based chemotherapy with a hazard ratio of 3.1 and a 95% CI of 1.2–7.7 ( p= 0.02). Conclusion. RhoC expression increases with breast cancer progression and RhoC protein level in tumor tissue is strongly associated with biologically aggressive invasive carcinomas of the breast. RhoC expression, if validated, may identify patients who are less likely benefit from doxorubicin therapy and suggests RhoC overexpression as a new target for intervention.

Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a 5-year disease-free survival of less than 45%. Little is known about the genetic alterations that result in IBC. In our previous work, we found that WISP3 was specifically lost in human IBC tumors when compared to stage-matched, non-IBC tumors. We hypothesize that WISP3 has tumor suppressor function in the breast and that it may be a key genetic alteration that contributes to the unique IBC phenotype. The full-length WISP3 cDNA was sequenced and cloned into an expression vector. The resulting construct was introduced in to the SUM149 cell line that was derived from a patient with IBC and lacks WISP3 expression. In soft agar, stable WISP3 transfectants formed significantly fewer colonies than the controls. Stable WISP3 transfectants lost their ability to invade and had reduced angiogenic potential. WISP3 transfection was effective in suppressing in vivo tumor growth in nude mice. Mice bearing WISP3 expressing tumors had a significantly longer survival than those with vector-control transfectant tumors. Our data demonstrate that WISP3 acts as a tumor suppressor gene in the breast. Loss of WISP3 expression contributes to the phenotype of IBC by regulating tumor cell growth, invasion and angiogenesis.

The most important factor in predicting outcome in patients with early breast cancer is the stage of the disease. There is no robust marker capable of identifying invasive carcinomas that despite their small size have a high metastatic potential, and that would benefit from more aggressive treatment. RhoC-GTPase is a member of the Ras-superfamily and is involved in cell polarity and motility. We hypothesized that RhoC expression would be a good marker to identify breast cancer patients with high risk of developing metastases, and that it would be a prognostic marker useful in the clinic. We developed a specific anti-RhoC antibody and studied archival breast tissues that comprise a broad spectrum of breast disease. One hundred eighty-two specimens from 164 patients were used. Immunohistochemistry was performed on formalin-fixed tissues. Staining intensity was graded 0 to 3+ (0 to 1+ was considered negative and 2 to 3+ was considered positive). RhoC was not expressed in any of the normal, fibrocystic changes, atypical hyperplasia, or ductal carcinoma in situ, but was expressed in 36 of 118 invasive carcinomas and strongly correlated with tumor stage (P = 0.01). RhoC had high specificity (88%) in detecting invasive carcinomas with metastatic potential. Of the invasive carcinomas smaller than 1 cm, RhoC was highly specific in detecting tumors that developed metastases. RhoC expression was associated with negative progesterone receptor and HER-2/neu overexpression. We characterized RhoC expression in human breast tissues. RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors smaller than 1 cm to guide treatment.

Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer known. IBC carries a guarded prognosis primarily due to rapid onset of disease, typically within six months, and the propensity of tumor emboli to invade the dermal lymphatics and spread systemically. Although the clinical manifestations of IBC have been well documented, until recently little was known about the genetic mechanisms underlying the disease. In a comprehensive study aimed at identifying the molecular mechanisms responsible for the unique IBC phenotype, our laboratory identified overexpression of RhoC GTPase in over 90% of IBC tumors in contrast to 36% of stage-matched non-IBC tumors. We also demonstrated that overexpression of RhoC GTPase in human mammary epithelial (HME) cells nearly recapitulated the IBC phenotype with regards to invasion, motility and angiogenesis. In the current study we sought to delineate which signaling pathways were responsible for each aspect of the IBC phenotype. Using well-established inhibitors to the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways. We found that activation of the MAPK pathway was responsible for motility, invasion and production of angiogenic factors. In contrast, growth under anchorage independent conditions was dependent on the PI3K pathway.

To realize the promise of precision medicine, it is important to integrate phenotypic assessment of cell populations to genomic data. The analysis of invading leader cells at the tumor invasion front is of interest as they may be guided by a targetable molecular phenotype. However, there is a lack of suitable platforms on which to analyze the tumor invasion front. In this study, we have designed and constructed a fluidic device for long-term (several days to weeks) 3-dimensional tumoroid culture of diverse cancer cells. Using this device, we can recapitulate the tumor invasion front and at the same time quantify the invasive potential of different breast cancer cell line models. Analyses of the tumor invasion front indicated a region of higher proliferation and suggest that the leader cells possess a different molecular phenotype from the tumoroid mass. Interestingly, significant heterogeneity among invading cells was still observed, suggesting that there could be: 1) the presence of multiple subpopulations of invasive cells, each with a different clonal genetic signature; and/or 2) reversible phenotypic switching occurring among invading cells due to phenotypic plasticity. These results obtained using this innovative device highlight and present a promising solution to the challenges developing adequate therapeutics accounting for tumor phenotypic heterogeneity. There is potential for the device for use in personalized medicine at diagnosis, allowing for both the quantification of disease progression risk as well as the molecular characterization of the invasive subpopulations from patient samples and their response to tailored therapies. Citation Format: Koh Meng Aw Yong, Christopher Oliver, Megan Altemus, Zhi Fen Wu, Sofia Merajver. Modeling the tumor invasion front using 3D fluidic tumoroid culture of cancer cells [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 5769. doi:10.1158/1538-7445.AM2017-5769

A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol-equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyplal-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O-depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal-tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic responsesmore » in vivo.« less

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.

To study the influence of transfection with human transforming growth factor-beta1 (hTGF-beta1) gene on the osteogenic potential and differentiation of the cultured human gingival fibroblast (GF).Enzyme kinetics method was used to measure the effects of the transfection on the alkaline phosphatase (ALP) activity. Immunohistochemistry stain and image analysis were applied to evaluate the alteration of the content of osteopontin (OPN), bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC) in the GF with transfection. Mineralization nodules formation in vitro was also used.The ALP activity of the GF after transfection was higher than the GF without transfection significantly (P0.05), and was close to that of the PDLCs (P0.05). The content of OC in GF was not improved after transfection, was similar with that of PDLCs (P0.05). Under immunohistochemistry stain, the contents of OPN, ON, BSP expressed in GF with transfection were higher than those of GF without transfection (P0.05), but similar to those of PDLCs (P0.05). In the mineralized cultured medium, the nodules were observed in the GF with transfection and PDLCs after 21 days and 24 days alternatively. After von Kossa stain, purple mineralization nodules were observed.The GF transfected with pcDNA3-hTGF-beta1 could express some osteogenic characters, though these characters are restricted.

To evaluate the effects of gingival fibroblasts (GF) transfected with hTGF-beta1 gene on improving the periodontal tissue regeneration for the repair of degree II artificial furcation defects.The gingival fibroblasts transfected with hTGF-beta1 gene was compounded to the cuttlebone-transformed nanometer hydroxyapatite (CBHA) material from the cuttlefish in vitro, the degree II furcation defects on the premolars of dogs were produced surgically, and the compound was to implanted into the defect (transfected group), and compared with the compound of periodontal ligament cells (PDLC) with nanometer HA material and the compound of untransfected GF with HA. The results were examined histologically 8 weeks after operation.In the transfected group and the positive control group, more new attachment was found compared with the negative control (P0.01), and the NC, NB and NC of the transfected group and the positive control group were: (2.97 +/- 0.50), (4.29 +/- 0.26) and (4.73 +/- 0.06) mm; (3.09 +/- 0.26), (4.46 +/- 0.25) and (4.69 +/- 0.10) mm, respectively. There was no significant difference between the two groups (P0.05). Although the alveolar bone regeneration was found in the untransfected group [NB = (3.46 +/- 0.32) mm], the root resorption was observed. The tracing experiment showed that the transfected GF were found in the new alveolar bone and the periodontal membrane.GF transfected with hTGF-beta1 gene can significantly improve the periodontal tissue regeneration in treatment of degree II furcation defects and is involved in the formation of the new alveolar bone and the new periodontal membrane.

Objective: In this study, the effects of baicalin – a flavonoid purified from Scutellaria baicalensis. Georgi, Scutellaria L. – on alveolar bone resorption in rat experimental periodontitis were examined. Method: 12 Sprague-Dawley rats (SD rats) were randomly divided into four groups: Group A1, A2, B and C. Except for Group C – the control group–, Group A1, A2 and B were used to establish the rat periodontitis model by repeat injection of Lipopolysaccharide (LPS). At the same time, Group A1 and A2 were injected with baicalin of different concentration. 9 days later, the maxillae were extracted and analyzed using micro computerized tomography (micro-CT), followed by histological analysis. Result: Micro-CT images showed that alveolar bone resorption was severely induced around the molar by repeat injection of LPS. Treatment with high-dose baicalin (1.0 μg / ml) clearly recovered alveolar bone resorption, meanwhile, low-dose baicalin(0.1 μg / ml) showed a similar but weaker effect. Histological examination clarified that the number of osteoclast was dose dependently decreased by baicalin treatment. Conclusion: These findings suggest that baicalin may inhibit the alveolar bone loss in periodontitis effectively.

To observe the effect of the self-made chitosan thermosensitive hydrogel system with dual-release bone morphogenetic protein and chlorhexidine on periodontal defects repair.The furcation defect model was established on dog premolar. The models were divided into five groups, including three experimental groups, one control group and one blank control group. The hydrogel with the chlorhexidine/3-cyclodextrin inclusion complexes (IC) /rhBMP-2, hydrogel with rhBMP-2, hydrogel with IC, the pure hydrogel were applied to the defects of the four groups, respectively, and the blank control group did not receive any agent. The dogs were sacrificed 8 weeks later and the periodontal regeneration and gingival condition were observed by histological examination.Obvious periodontal tissue regeneration was found in group one and two. The heights of new bone reached 99.2% of the defects in group one, 87.8%, 63.6%, 37.0% and 34.3% in group two, three, four and blank control groups, respectively. The inflammation of the affected gingiva showed less significant in group one and group three than in the other groups.rhBMP-2 and chlorhexidine played their independent role in repairing periodontal defects and the dual-release chitosan thermosensitive hydrogel system is effective and convenient to use.

To observe the effect of the chitosan thermosensitive hydrogel loading recombinant human bone morphogenetic protein-2 (rhBMP-2) on repairing periodontal defects.To prepare artificial furcation defects model in the posterior area in 3 healthy male dogs, and then to inject chitosan thermosensitive hydrogel loading of rhBMP-2 after fast suturing tissue flap. The groups filled with nothing or filled only with chitosan thermosensitive hydrogel were the controls. The dogs were sacrificed after 5 weeks and the periodontal regeneration was observed histologically.The histological observation showed that the chitosan thermosensitive hydrogel loading rhBMP-2 group achieved apparent periodontal tissue regeneration occupying the majority of the defects and the control groups got only a small amount of periodontal tissue regeneration.The chitosan thermosensitive hydrogel loading rhBMP-2 can effectively promote the periodontal tissue regeneration, while simplifying the surgical operation. It might be a potential means for periodontal regeneration.

The chitosan thermosensitive hydrogel is liquid at room temperature but gels rapidly when heated to body temperature. This hydrogel are wildly used for cell encapsulation, drug delivery or tissue-engineered scaffolds. The system can sustain the release of macromolecules over a period of several hours to a few days. However, with low-molecular-weight compounds, the release is generally completed within 24 h. To prepare a functional chitosan thermosensitive hydrogel for slow release both broad-spectrum antibiotic chlorhexidine and growth factor recombined human bone morphogenetic protein-2 (rhBMP-2), The beta-cyclodextrin was used to prepare an inclusion complex with chlorhexidine, and then the latter was incorporated into the chitosan thermosensitive hydrogel system. Simultaneously, rhBMP-2 was added into the hydrogel system. By HAAKE viscosity measuring instrument, we contrasted the viscoelastic properties of system with or without objective factors. And the in vitro release kinetics of chlorhexidine and rhBMP-2 was investigated by HPLC (high performance liquid chromatography) and ELISA (enzyme-linked immunosorbent assay) respectively. The results showed that the addition of chlorhexidine/beta-cyclodextrin inclusion complex to the thermosensitive solution did not change the gelling behavior of the thermosensitive system. Further, the in vitro release profiles demonstrated that the release rate of chlorhexidine and rhBMP-2 from hydrogel became slower, controlled delivery over at least 1 month. By first preparing chlorhexidine/beta-cyclodextrin inclusion complex, and then mixing the IC and rhBMP-2 into the chitosan thermosensitive hydrogel, a functional chitosan thermosensitive hydrogel system with ability of slow release both rhBMP-2 and chlorhexidine is successfully made.

Objective: The aim of this work was to estimate the present periodontal problems of people in China, based on an epidemiological investigation of adults. Material and Methods: The data were collected from the northwest, southwest, northeast and east regions (400 subjects from each region) of China. All subjects were over 25 years of age. About half of the subjects were farmers and about half were urban professionals. Everyone was asked to fill out a questionnaire and to undergo a professional oral examination. Periodontal health status was evaluated by a simplified oral hygiene index (OHI-S), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PD), clinical attachment loss (CAL), and tooth mobility. Results: Of the 1590 subjects enrolled in this investigation, 45.7% were male, 45.5% were farmers, and the remaining were urban professionals, and 27.7% of the subjects were smokers. There was a significant difference in the educational background but not smoking between the rural and urban groups. While 34.9% of the subjects in the urban group brushed only once per day, 56.1% of the subjects in the rural group did so. The prevalence of bleeding during brushing was 71.1%, while about 61.4% of the subjects know nothing about scaling. All periodontal indices were significantly higher in males than in females and higher in the rural group than in the urban group. PD, CAL and tooth mobility increased with age. The percentage of sites with CAL>3 mm in the rural group (49.5%) was significantly higher than that in the urban group (37.5%). Both current and former smokers showed increased CAL than non-smokers. Conclusion: Gingivitis and periodontitis are common findings in China. Most Chinese have no knowledge of common periodontal prevention and treatment and very few have regular dental care. The data of this study suggest that age, smoking, and limited education are significantly associated with Chinese adult periodontal attachment loss. Preventive periodontal care and education should be reinforced in the future by establishing relevant oral health projects.

To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene.pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT.IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation.The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.

To prepare enamel matrix proteins (EMPs) loaded dextran-based hydrogel microspheres (EMPs-dex-MPs), and to evaluate their EMPs controlled release property and their biological effects on the proliferation and differentiation of human periodontal ligament cells (PDLCs) in vitro.Using dimethylbenzene as the oil phase, EMPs-dex-MPs were achieved by emulsion-chemical crosslinking technique. The process of the recombination preparation was optimized by orthogonal factorization method. The configuration and size of EMPs-dex-MPs were determined by scanning electron microscope. The EMPs loading content and encapsulation rate of EMPs-dex-MPs, and their biodegradation characteristic were studied by routine analysis methods. Dynamic dialysis method was used to determine the release characteristic of EMPs-dex-MPs in vitro and its influencing factors. The proliferation of cultured PDLCs was measured by MTF method and the differentiation of PDLCs was measured by their alkaline phosphatase (ALP) activities.The results showed that EMPs-dex-MPs were homogenous and stable with the average diameter 25 microm, and the EMPs loading content was (32.8 +/- 1.2)%, the encapsulation rate was (78.9 +/- 1.0)%. Under 9% physiological saline solution contained a very thimbleful quantity of dextranase EMPs-dex-MPs could be biodegraded completely during about 40 days. The in vitro experiments showed that about 80% of EMPs could be released out in 20 days. Using EMPs-dex-MPs could enhance the proliferation responses and ALP activities of PDLCs more than 12 days.As a new sustained release system of growth factors, the dex-MPs is stable, workable and biodegradable. EMPs-dex-MPs, whose drug release can be controlled by preparation technique, may be more effective in promoting periodontal tissue regeneration.

To prepare and study the recombinant human bone morphogenetic protein-2 loaded dextran-based hydrogel nanospheres (rhBMP(2)-dex-NPs) sustained release system, and to evaluate its biological effects on cultured rabbit bone mesenchymal stem cells(BMSCs).The rhBMP(2)-dex-NPs were prepared by improved emulsion polymerization method. Their morphology, size and size distribution, encapsulated ratio and stability were assessed by routine procedure. Dynamic dialysis method was used to determine the release characteristics of rhBMP(2)-dex-NPs in vitro. Cell culture technique and MTT colorimetric assay were used to evaluate the proliferation and differentiation of the BMSCs, ALP kit was used to evaluate the ALP activity of the BMSCs so as to show the differentiation of the cells by adding the rhBMP(2)-dex-NPs to the DMEM culture medium (B group) or rhBMP2 only (A group). Adding dex-NPs without rhBMP2 (C group) and adding nothing (D group) were taken as the controls. The results were analyzed by statistical analysis software (SPSS10.0).The shape of rhBMP(2)-dex-NPs was spherical, with a size distribution of 20 nm. The encapsulated ratio was 83% and rhBMP(2)-dex-NPs could be kept more than 6 months under 4 degrees C without decomposition , destruction or deposition. The release profile in vitro was in accordance with two phases kinetics law, and more than 80% of the encapsulated rhBMP(2) can be released during 12 days. Statistical analysis showed that rhBMP(2)-dex-NPs had biological activity, and could enhance both proliferation and differentiation of rabbit BMSCs significantly, the effect of the rhBMP(2)-dex-NPs was significantly higher than that of rhBMP(2) (P0.01). During the first 3 days, the proliferation and differentiation of BMSCs between group A and B had no significance (P0.05), but much faster than group C and D. After 5 to 7 days, rhBMP(2)-dex-NPs could enhance BMSCs proliferation and differentiation continually, but rhBMP2 had no enhancement any more. 7 days later, the difference between group A and B become much more significant (P0.001).The rhBMP(2)-dex-NPs can release rhBMP2 more than 12 days and have long-drawn biological effects. To encapsulate rhBMP2 into dextran-based hydrogel nanospheres may be an effective way of growth factor controlled release in tissue engineering.

To observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.PcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.The results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

To observe the effects of cells and scaffolds tissue engineering on the periodontal regeneration, and to evaluate the feasibility of nano-Hap-collagen (nHAC) as the scaffold material for periodontal tissue engineering.Dog autogenous periodontal ligament cells (PDLCs) cultured in vitro were collected and seeded on the three-dimensional framework of nHAC. The cell growth in the scaffolds was observed by scanning electron microscope. And then the PDLCs-nHAC composites were transplanted into man-made periodontal defects, and the groups filled with nothing or filled only with nHAC were the controls. The dogs were sacrificed after 8 weeks and the periodontal regeneration was observed histologically.Scanning electron microscope showed the porous structure of nHAC and the eugonic growth of cells in the nHAC scaffolds. The histological observation showed that the PDLCs-nHAC groups exhibited more new bone, new periodontal ligament and new cementum occupying the majority of the defects than the control groups, and the epitheliums were not observed.Periodontal regeneration could be enhanced by the cells and scaffolds tissue engineering, and the PDLCs and nHAC could be used as the seed cell and the scaffold material for periodontal tissue engineering.

Inflammatory breast cancer (IBC) is the most deadly form of breast cancer in humans presumably due to its ability to metastasize from its inception. In our laboratory, overexpression of RhoC GTPase was observed to be specific for IBC tumors, but not for stage-matched, non-IBC tumors. RhoC is known to contribute to an IBC-like phenotype in HPV-E6E7 immortalized breast cells. To further study the effect of RhoC overexpression on IBC metastasis, we generated stable transfectants of spontaneous immortalized mammary epithelial cells (MCF10A) overexpressing wild-type RhoC or a constitutively active RhoC mutant (G14V). Both the RhoC wild type and the G14V transfectants were highly invasive and proliferated more rapidly compared to vector-only control clones. Overexpression of RhoC led to an increase in actin stress fiber and focal adhesion contact formation. Comparative microarray analysis of these clones further revealed that RhoC overexpression upregulated 108 genes whereas seven genes were down-regulated. We have further verified by quantitative RT-PCR that genes involved in cell proliferation, invasion/adhesion, and angiogenesis were modulated by RhoC. This work suggests strong candidates for the downstream oncogenic functions of RhoC.

Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer known. IBC carries a guarded prognosis primarily due to rapid onset of disease, typically within six months, and the propensity of tumor emboli to invade the dermal lymphatics and spread systemically. Although the clinical manifestations of IBC have been well documented, until recently little was known about the genetic mechanisms underlying the disease. In a comprehensive study aimed at identifying the molecular mechanisms responsible for the unique IBC phenotype, our laboratory identified overexpression of RhoC GTPase in over 90% of IBC tumors in contrast to 36% of stage-matched non-IBC tumors. We also demonstrated that overexpression of RhoC GTPase in human mammary epithelial (HME) cells nearly recapitulated the IBC phenotype with regards to invasion, motility and angiogenesis. In the current study we sought to delineate which signaling pathways were responsible for each aspect of the IBC phenotype. Using well-established inhibitors to the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways. We found that activation of the MAPK pathway was responsible for motility, invasion and production of angiogenic factors. In contrast, growth under anchorage independent conditions was dependent on the PI3K pathway.

The most important factor in predicting outcome in patients with early breast cancer is the stage of the disease. There is no robust marker capable of identifying invasive carcinomas that despite their small size have a high metastatic potential, and that would benefit from more aggressive treatment. RhoC-GTPase is a member of the Ras-superfamily and is involved in cell polarity and motility. We hypothesized that RhoC expression would be a good marker to identify breast cancer patients with high risk of developing metastases, and that it would be a prognostic marker useful in the clinic. We developed a specific anti-RhoC antibody and studied archival breast tissues that comprise a broad spectrum of breast disease. One hundred eighty-two specimens from 164 patients were used. Immunohistochemistry was performed on formalin-fixed tissues. Staining intensity was graded 0 to 3+ (0 to 1+ was considered negative and 2 to 3+ was considered positive). RhoC was not expressed in any of the normal, fibrocystic changes, atypical hyperplasia, or ductal carcinoma in situ, but was expressed in 36 of 118 invasive carcinomas and strongly correlated with tumor stage (P = 0.01). RhoC had high specificity (88%) in detecting invasive carcinomas with metastatic potential. Of the invasive carcinomas smaller than 1 cm, RhoC was highly specific in detecting tumors that developed metastases. RhoC expression was associated with negative progesterone receptor and HER-2/neu overexpression. We characterized RhoC expression in human breast tissues. RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors smaller than 1 cm to guide treatment.

It is increasingly likely that the high incidence of off-target effects associated with targeted inhibitors is due, in part, to the highly complex and dysregulated intracellular molecular networks associated with cancer. Ignoring this complexity can lead to suboptimal results and subsequent loss of life through ineffective therapies. The phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways are two of the most dysregulated pathways across all cancers. Several regulatory mechanisms have been proposed to explain the apparent cross-talk between them. For example, RAS to ERK signaling in the MAPK pathway has been proposed as an important metastases “escape mechanism” when PI3K is inhibited. In order to rationally develop precise therapeutic avenues to target these oncogenic pathways, it is critical to understand how the pathways are wired as an integrated network, in both normal and tumor cells. We have developed a systems biology approach that integrates measurements of protein activation under diverse experimental conditions, including inhibition of MEK and PI3K, with a novel network inference computational model that predicts the causal connectivity of a network from experimental data. The network inference model utilizes a genetic algorithm to search for the “optimal” network configuration that most closely matches the experimental data used as input. We validated the approach using in silico data generated from a set of randomized test networks. Next, we applied this approach to breast epithelial cell lines (MFC10A, MCF7, MDA-MB-231, and SUM149) by performing a set of experiments using a series of pathway specific inhibitors with and without growth factor stimulation. From phospho western blot readouts of several proteins in the PI3K and MAPK pathways, we have predicted network configurations most likely responsible for the distinct experimental output of each of the four cell lines. In some cases, our network inference model predicted multiple optimal networks for a given cell line. Our method predicted the next experiments needed to optimally distinguish between the set of possible candidate networks. Our results suggest that some proposed interactions and feedback mechanisms attributed to MAPK and PI3K cross-talk in the literature may not be valid. This approach has important implications for the prospect of effective personalized cancer treatments and targeted molecular inhibition. Elucidating the mechanisms of cross-talk between the MAPK and PI3K pathways in cells collected from patient tumors will permit rational discovery of the optimal combination of targeted therapies needed to treat a specific cancer, based on the actual network that is operational in the tumor. Moreover, our methodology and predictive network model can be applied to any set of signaling pathways. Citation Format: Megan E. Egbert, Michelle L. Wynn, Zhi Fen Wu, Rabia A. Gilani, Santiago Schnell, Sofia D. Merajver. A systems biology approach for rational molecular network inference. [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 357. doi:10.1158/1538-7445.AM2014-357

Targeted molecular inhibitors have emerged as a leading anti-cancer strategy; however, despite promising pre-clinical data, many targeted inhibitors induce undesirable off-target effects in the clinic. The large number of off-target effects associated with molecular inhibitors was recently termed the ‘‘whack a mole problem’’ because inhibiting one molecular target often unintentionally activates another molecule. It is increasingly clear that the high incidence of off-target effects associated with targeted inhibitors is related to the complex interactions and emergent behaviors inherent to the highly complex and dysregulated intracellular networks of cancer. Both the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways are known to be dysregulated in cancer. In previous work, we and others have demonstrated that the MAPK pathway promotes motility, invasion, and angiogenic factors while the PI3K pathway plays an important role in controlling anchorage independent growth. In addition, the PI3K pathway plays an essential role in stimulating glucose metabolism and the Warburg effect. We hypothesize that robust interactions exist between these two pathways that influence efficacy and potentially also acquired resistance to targeted therapies. Using a combination of experimental and theoretical techniques, we developed a predictive network model linking growth factor signaling to the MAPK and PI3K pathways as well as to glucose metabolism. Specifically, we constructed a logic-based network of the cross-talk between MAPK and PI3K signaling that relied on a detailed literature survey to identify known molecular interactions as well as proposed interactions and regulatory feedback connections in the literature. We next performed a set of experiments using a normal-like breast epithelial cell line and a series of pathway specific inhibitors with and without growth factor stimulation to validate our model. Finally, we repeated these experiments using a diverse set of breast cancer cell lines and integrated this data to produce a series of cancer networks representative of different stages of breast cancer progression. Our model was able to recapitulate both our own experimental data and published data in the literature using a smaller subset of regulatory feedback mechanisms than we started with. Together, our results suggest that some proposed interactions and feedback mechanisms attributed to MAPK and PI3K cross-talk in the literature may not be valid. Citation Format: Megan E. Egbert, Michelle L. Wynn, Zhi Fen Wu, Santiago Schnell, Sofia D. Merajver. Elucidating the complex cross-talk between the MAPK and PIK3 pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2013-5223

Cancer cells exhibit a metabolic phenotype characterized by high rates of glucose uptake and lactate production, known as the Warburg effect. While the Warburg effect and normal proliferative metabolism appear similar, important molecular differences exist. We hypothesize that molecular and metabolic drivers of the Warburg effect can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Intracellular networks exhibit a variety of emergent non-linear behaviors and, as a result, the use of experimental intuition alone will not be enough to identify these drivers. Using a combination of experimental and theoretical methods, we developed a model of breast cancer progression that includes metabolism and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, an important regulator of carbon metabolism. A key component of our model is a detailed logic network of molecular interactions associated with PI3K signaling as well as regulatory connections to central carbon metabolism, including the ATP/AMP ratio, GLUT receptor activation, hexokinase activation, and changes in the catalytic activity of pyruvate kinase. To validate our model, a series of phospho Western blot analyses were performed using a normal-like breast cell line and a diverse set of breast cancer cell lines exposed to PI3K pathway inhibitors. From these data, a series of predictive network models were constructed representing distinct stages of breast cancer progression. We also generated detailed metabolic flux maps for each cell line using metabolic flux analysis (MFA), a method that relies on carbon-13 tracers, mass-spectrometry, and measurements of extracellular flux to infer intracellular flux. In agreement with recent studies, we found an increase in the reductive carboxylation of glutamine derived alpha-ketoglutarate in cells constitutively adapted to hypoxia. We also identified a potentially important metabolic vulnerability in aggressive breast cancers. Moreover, we found important PI3K network differences at the RNA and protein levels, some of which were isoform specific. Together our data indicate that very different system-level properties are associated with distinct stages of breast cancer progression and metabolic transformation. Our model is suitable for performing in silico molecular perturbations to predict a normal as well as tumor level response to a targeted therapy or combination of therapies. Our approach also serves as a prototype for the use of systems biology methods in personalized medicine where molecular and metabolic data collected from a patient's biopsied tumor is input into a predictive model designed to develop a strategic treatment plan for the patient. The use of predictive models to integrate data from an individual patient will have a profound impact on cancer care decisions and patient outcomes in the future. Citation Format: Michelle L. Wynn, Megan Egbert, Lauren D. Van Wassenhove, Zhi Fen Wu, Firas Midani, Charles Evans, Charles F. Burant, Santiago Schnell, Sofia D. Merajver. Unraveling the complex regulatory relationship between PI3K signaling and metabolic transformation in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5239. doi:10.1158/1538-7445.AM2013-5239

Background Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. We found concordant and consistent alterations of two genes in 90% of IBC tumors when compared with stage-matched non-IBC tumors: overexpression of RhoC guanosine triphosphatase and loss of WNT-1 induced secreted protein 3 (WISP3). Further work revealed that RhoC is a transforming oncogene for human mammary epithelial (HME) cells. Despite the aggressiveness of the RhoC-driven phenotype, it does not quantitatively reach that of the true IBC tumors. We have demonstrated that WISP3 has tumor growth and angiogenesis inhibitory functions in IBC. We proposed that RhoC and WISP3 cooperate in the development of IBC.