Malathy P.V. Shekhar

Host microenvironment in breast cancer development: Extracellular matrix–stromal cell contribution to neoplastic phenotype of epithelial cells in the breast

Epithelial–mesenchymal interactions play an important role both in normal mammary gland development and during neoplastic transformation. Perturbations in the production, deposition and degradation of the extracellular matrix occurring during neoplastic transformation and progression have been implicated to arise from alterations in the stromal response. These changes in the stroma exhibit a dominant regulatory role, via microenvironmental epigenetic effectors, to contribute to the development of the tumorigenic epithelial phenotype. The role of stromally derived microenvironmental epigenetic effectors in modulating epithelial growth, hormonal response, morphogenesis and epithelial plasticity is discussed.

Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance

Tumor drug resistance significantly limits the success of chemotherapy in the clinic. Tumor cells utilize multiple mechanisms to prevent the accumulation of anticancer drugs at their intracellular site of action. In this study, we investigated the anticancer efficacy of doxorubicin in combination with photodynamic therapy using methylene blue in a drug-resistant mouse tumor model. Surfactant-polymer hybrid nanoparticles formulated using an anionic surfactant, Aerosol-OT (AOT), and a naturally occurring polysaccharide polymer, sodium alginate, were used for synchronized delivery of the two drugs. Balb/c mice bearing syngeneic JC tumors (mammary adenocarcinoma) were used as a drug-resistant tumor model. Nanoparticle-mediated combination therapy significantly inhibited tumor growth and improved animal survival. Nanoparticle-mediated combination treatment resulted in enhanced tumor accumulation of both doxorubicin and methylene blue, significant inhibition of tumor cell proliferation, and increased induction of apoptosis. These data suggest that nanoparticle-mediated combination chemotherapy and photodynamic therapy using doxorubicin and methylene blue has significant therapeutic potential against drug-resistant tumors.

Alterations in Galectin-3 Expression and Distribution Correlate with Breast Cancer Progression : Functional Analysis of Galectin-3 in Breast Epithelial-Endothelial Interactions

To define the role of galectin-3 in breast cancer progression, we have used a novel three-dimensional co-culture system that recapitulates in vivo reciprocal functional breast epithelial-endothelial cell-cell and cell-matrix interactions, and examined the expression of galectin-3 mRNA and protein in human breast tumors and xenografts. Galectin-3 is required for the stabilization of epithelial-endothelial interaction networks because immunoneutralization with galectin-3 antibodies abolishes the interactions in a dose-dependent manner. Co-culture of epithelial cells with endothelial cells results in increase in levels of secreted galectin-3 and presence of proteolytically processed form of galectin-3 in the conditioned media. In contrast, intracellular galectin-3 predominantly exists in the intact form. This difference in sensitivity to proteolytic processing of secreted versus intracellular galectin-3 probably arises from differences in accessibility of protease-sensitive sites, levels, and/or type of activated protease(s), and may be indicative of different functional roles for intact and processed galectin-3. To determine whether the proteolytically cleaved galectin-3 retains its ability to bind to endothelial cells, binding assays were performed with the full-length and matrix metallopeoteinase-2-cleaved recombinant galectin-3. Although a dose-dependent increase in binding to human umbilical vein endothelial cells was observed with both full-length and cleaved galectin-3, proteolytically cleaved galectin-3 displayed approximately 20-fold higher affinity for human umbilical vein endothelial cells as compared to the full-length protein. Examination of galectin-3 expression in breast tumors and xenografts revealed elevated levels of galectin-3 mRNA and protein in the luminal epithelial cells of normal and benign ducts, down-regulation in early grades of ductal carcinoma in situ (DCIS), and re-expression in peripheral tumor cells as DCIS lesions progressed to comedo-DCIS and invasive carcinomas. These data suggest that galectin-3 expression is associated with specific morphological precursor subtypes of breast cancer and undergoes a transitional shift in expression from luminal to peripheral cells as tumors progressed to comedo-DCIS or invasive carcinomas. Such a localized expression of galectin-3 in cancer cells proximal to the stroma could lead to increased invasive potential by inducing novel or better interactions with the stromal counterparts.

Surfactant−Polymer Nanoparticles Enhance the Effectiveness of Anticancer Photodynamic Therapy

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

Genome based cell population heterogeneity promotes tumorigenicity: The evolutionary mechanism of cancer

Cancer progression represents an evolutionary process where overall genome level changes reflect system instability and serve as a driving force for evolving new systems. To illustrate this principle it must be demonstrated that karyotypic heterogeneity (population diversity) directly contributes to tumorigenicity. Five well characterized in vitro tumor progression models representing various types of cancers were selected for such an analysis. The tumorigenicity of each model has been linked to different molecular pathways, and there is no common molecular mechanism shared among them. According to our hypothesis that genome level heterogeneity is a key to cancer evolution, we expect to reveal that the common link of tumorigenicity between these diverse models is elevated genome diversity. Spectral karyotyping (SKY) was used to compare the degree of karyotypic heterogeneity displayed in various sublines of these five models. The cell population diversity was determined by scoring type and frequencies of clonal and non‐clonal chromosome aberrations (CCAs and NCCAs). The tumorigenicity of these models has been separately analyzed. As expected, the highest level of NCCAs was detected coupled with the strongest tumorigenicity among all models analyzed. The karyotypic heterogeneity of both benign hyperplastic lesions and premalignant dysplastic tissues were further analyzed to support this conclusion. This common link between elevated NCCAs and increased tumorigenicity suggests an evolutionary causative relationship between system instability, population diversity, and cancer evolution. This study reconciles the difference between evolutionary and molecular mechanisms of cancer and suggests that NCCAs can serve as a biomarker to monitor the probability of cancer progression. J. Cell. Physiol. 219: 288–300, 2009.

Diverse system stresses: common mechanisms of chromosome fragmentation

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Rad6B Is a Positive Regulator of β-Catenin Stabilization

Mutations in beta-catenin or other Wnt pathway components that cause beta-catenin accumulation occur rarely in breast cancer. However, there is some evidence of beta-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of beta-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces beta-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, beta-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus-Wnt-1 mammary tumor-derived cells, implicating Rad6B in physiologic regulation of beta-catenin stability and activity. Ubiquitinated beta-catenin was detectable in chromatin immunoprecipitations performed with beta-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of beta-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular beta-catenin but with minimal effects on cell membrane-associated beta-catenin. Measurement of beta-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular beta-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates beta-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of beta-catenin, reveal a positive regulatory feedback loop between Rad6B and beta-catenin and a novel mechanism of beta-catenin stabilization/activation in breast cancer cells.

Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response

ABSTRACT The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G2/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.

RAD6B overexpression confers chemoresistance: RAD6 expression during cell cycle and its redistribution to chromatin during DNA damage-induced response

The HR6A and HR6B genes, homologs of the yeast RAD6 gene, encode ubiquitin conjugating enzymes that are required for postreplication repair (PRR) of DNA and damage-induced mutagenesis. We show here that consistent with its role as a PRR protein, HR6 protein (referred as RAD6) expression is cell cycle regulated, with maximal levels expressed in late S/G2 phases of the cell cycle. Exposure of MCF10A cells to adriamycin (ADR) causes enhancement in the levels of RAD6B mRNA and protein. Inclusion of actinomycin D abolishes both basal and ADR-induced RAD6B transcription indicating that ADR-induced effects on RAD6B transcription result from an increase in transcriptional activity rather than from regulation of RAD6B mRNA stability. The increase in RAD6 protein expression observed in ADR-treated cells is dependent upon transcription and de novo protein synthesis, as addition of actinomycin D and cycloheximide eliminated the induction effects. Using in vivo crosslinking experiments, we demonstrate that only a small proportion of RAD6 is associated with chromatin in untreated MCF10A cells. However, treatment with ADR or cisplatin is accompanied by a significant increase and redistribution of RAD6 to DNA, and RAD6, RAD18, PCNA, phosphohistone H3, as well as p53 proteins are all found in the DNA fractions. These findings suggest that although RAD6 protein is present in the nucleus, its recruitment to the chromatin appears to be modulated by DNA damage. Whereas MCF10A cells engineered to overexpress ectopic RAD6B are significantly more resistant to ADR and cisplatin as compared to empty vector-transfected cells, MCF10A cells stably transfected with antisense RAD6B display hypersensitivity to these damage-inducing drugs. Analysis of PRR capacities in cisplatin-treated MCF10A cells stably transfected with empty vector, RAD6B or antisense RAD6B showed that whereas RAD6B-overexpressing and vector control MCF10A cells possessed the ability to convert newly synthesized DNA to higher molecular weight species, MCF10A cells depleted of RAD6B are PRR-compromised. Although no human diseases have been linked to mutations in the PRR pathway genes, these data suggest that RAD6 may play an essential role in DNA damage tolerance and recovery via modulation of PRR, and that imbalances in the levels of RAD6 could lead to changes in drug sensitivity and damage-induced mutagenesis.

Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance.

Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cells enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed.