RAD6B Loss Disrupts Expression of Melanoma Phenotype in Part by Inhibiting WNT/ß-Catenin Signaling.

Canonical Wnt signaling is critical for melanocyte lineage commitment and melanoma development. RAD6B, a ubiquitin-conjugating enzyme critical for translesion DNA synthesis, potentiates ß-catenin stability/activity by inducing proteasome-insensitive polyubiquitination. RAD6B expression is induced by ß-catenin, triggering a positive feedback loop between the two proteins. RAD6B function in melanoma development/progression was investigated by targeting RAD6B using CrispR/Cas9 or an RAD6-selective small-molecule inhibitor #9 (SMI#9). SMI#9 treatment inhibited melanoma cell proliferation but not normal melanocytes. RAD6B knockout or inhibition in metastatic melanoma cells downregulated ß-catenin, ß-catenin-regulated microphthalmia-associated transcription factor (MITF), sex-determining region Y-box 10, vimentin proteins, and MITF-regulated melan A. RAD6B knockout or inhibition decreased migration/invasion, tumor growth, and lung metastasis. RNA-sequencing and stem cell pathway real-time RT-PCR analysis revealed profound reductions in WNT1 expressions in RAD6B knockout M14 cells compared with control. Expression levels of ß-catenin-regulated genes VIM, MITF-M, melan A, and TYRP1 (a tyrosinase family member critical for melanin biosynthesis) were reduced in RAD6B knockout cells. Pathway analysis identified gene networks regulating stem cell pluripotency, Wnt signaling, melanocyte development, pigmentation signaling, and protein ubiquitination, besides DNA damage response signaling, as being impacted by RAD6B gene disruption. These data reveal an important and early role for RAD6B in melanoma development besides its bonafide translesion DNA synthesis function, and suggest that targeting RAD6B may provide a novel strategy to treat melanomas with dysregulated canonical Wnt signaling.

RAD6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by aggravating mitotic spindle damage.

BACKGROUND: Paclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation. METHODS: TNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin. RESULTS: Our data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity. CONCLUSIONS: These data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

Gold nanoparticle conjugated Rad6 inhibitor induces cell death in triple negative breast cancer cells by inducing mitochondrial dysfunction and PARP-1 hyperactivation: Synthesis and characterization.

We recently developed a small molecule inhibitor SMI#9 for Rad6, a protein overexpressed in aggressive breast cancers and involved in DNA damage tolerance. SMI#9 induces cytotoxicity in cancerous cells but spares normal breast cells; however, its therapeutic efficacy is limited by poor solubility. Here we chemically modified SMI#9 to enable its conjugation and hydrolysis from gold nanoparticle (GNP). SMI#9-GNP and parent SMI#9 activities were compared in mesenchymal and basal triple negative breast cancer (TNBC) subtype cells. Whereas SMI#9 is cytotoxic to all TNBC cells, SMI#9-GNP is endocytosed and cytotoxic only in mesenchymal TNBC cells. SMI#9-GNP endocytosis in basal TNBCs is compromised by aggregation. However, when combined with cisplatin, SMI#9-GNP is imported and synergistically increases cisplatin sensitivity. Like SMI#9, SMI#9-GNP spares normal breast cells. The released SMI#9 is active and induces cell death via mitochondrial dysfunction and PARP-1 stabilization/hyperactivation. This work signifies the development of a nanotechnology-based Rad6-targeting therapy for TNBCs. FROM THE CLINICAL EDITOR: Protein Rad6 is overexpressed in breast cancer cells and its blockade may provide a new treatment against 3N breast cancer. The authors conjugated a small molecule inhibitor SMI#9 for Rad6 to gold nanoparticles in this study and showed that this new formulation specifically targeted chemo-resistant breast cancer cells and highlighted the importance of nanotechnology in drug carrier development.

Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.

The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces ß-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify ß-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in ß-catenin, and characterize their breast cancer tissue expression. ß-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-ß-catenin and His-Rad6B deletion mutants identified amino acids 131-181 and 50-116, respectively, as necessary for their interaction. Ubiquitination assays using ß-catenin deletion mutants mapped Rad6B-induced ubiquitination within ß-catenin 181-422 encompassing Armadillo repeats 2-7. Lysine to arginine mutations within repeats 5-7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a ß-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-ß-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-ß-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a ß-catenin/TCF transcriptional target, was also reduced in K394R-ß-catenin transfected cells. Steady-state K394R-ß-catenin levels are decreased compared to wild type-ß-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated ß-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of ß-catenin ubiquitination that may be important for the stability and activity of ß-catenin in breast cancer.

Design, synthesis and in vitro anticancer evaluation of 4,6-diamino-1,3,5-triazine-2-carbohydrazides and -carboxamides.

Series of substituted 4,6-diamino-1,3,5-triazine-2-carbohydrazides and -carboxamides have been synthesised, based on molecular modelling of candidate structures related to the previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate anticancer agents TZ8 and TZ9. Synthesis of the target compounds was readily accomplished in two steps from aryl biguanides via reaction of phenylhydrazine or benzylamines with key 4-amino-6-(arylamino)-1,3,5-triazine-2-carboxylate intermediates. These new triazine derivatives were tested for in vitro anticancer activity against the Rad6B expressing human breast cancer cell lines MDA-MB-231 and MCF-7. Active compounds, such as the triazinyl-carbohydrazides 3a-e, were found to exhibit low micromolar IC50 values particularly in the Rad6B-overexpressing MDA-MB-231 cell line.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

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.

Alternative Splicing of RAD6B and Not RAD6A is Selectively Increased in Melanoma: Identification and Functional Characterization.

Rad6B, a principal component of the translesion synthesis pathway, and activator of canonical Wnt signaling, plays an essential role in cutaneous melanoma development and progression. As Rad6 is encoded by two genes, namely, UBE2A (RAD6A) and UBE2B (RAD6B), in humans, we compared their expressions in melanomas and normal melanocytes. While both genes are weakly expressed in normal melanocytes, Rad6B is more robustly expressed in melanoma lines and patient-derived metastatic melanomas than RAD6A. The characterization of RAD6B transcripts revealed coexpression of various splice variants representing truncated or modified functional versions of wild-type RAD6B in melanomas, but not in normal melanocytes. Notably, two RAD6B isoforms with intact catalytic domains, RAD6BΔexon4 and RAD6Bintron5ins, were identified. We confirmed that RAD6BΔexon4 and RAD6Bintron5ins variants are expressed as 14 and 15 kDa proteins, respectively, with functional in vivo ubiquitin conjugating activity. Whole exome sequence analysis of 30 patient-derived melanomas showed RAD6B variants coexpressed with wild-type RAD6B in all samples analyzed, and RAD6Bintron5ins variants were found in half the cases. These variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly wild-type. The expression of functional RAD6B variants only in melanomas reveals RAD6B's molecular heterogeneity and its association with melanoma pathogenesis.

Erratum: Functional analysis of MKP-1 and MKP-2 in breast cancer tamoxifen sensitivity.

[This corrects the article DOI: 10.18632/oncotarget.1795.].

Essential role of T-cell factor/beta-catenin in regulation of Rad6B: a potential mechanism for Rad6B overexpression in breast cancer cells.

We have previously shown that the postreplication DNA repair gene Rad6B plays a critical role in the maintenance of genomic integrity of human breast cells. Whereas normal breast cells express low levels of Rad6B, increases in Rad6B expression occur in hyperplasia with overexpression in breast carcinomas. Here, we show that the human Rad6B gene is a transcriptional target of T-cell factor (TCF)-4/beta-catenin/p300. Rad6B promoter activity is subject to negative regulation in normal human MCF10A breast cells whereas it is constitutively active in metastatic MDA-MB-231 breast cancer cells. Derepression and activation of Rad6B promoter in MCF10A cells require coexpression of beta-catenin and p300. Using electrophoresis mobility shift assay, Western blot analysis of electrophoresis mobility shift assay, UV cross-linking, and chromatin immunoprecipitation assay, we show that Rad6B transcriptional repression in MCF10A cells is due to paucity of transcriptionally active beta-catenin assembled on the TCF binding sequence in the Rad6B promoter rather than to a deficit/decreased affinity of TCF-4 for the TCF binding element in Rad6B promoter. Three-dimensional epithelial acini generated in vitro from MCF10A cells cotransfected with beta-catenin and p300 showed beta-catenin expression on the membrane, cytoplasm, and/or nuclei with concomitant Rad6 overexpression, whereas control acini showed beta-catenin on the membranes and negligible Rad6 expression. Immunohistochemical analysis of 12 breast carcinomas showed an approximately 80% correlation between Rad6 and beta-catenin expression, and combined nuclear and cytoplasmic staining of beta-catenin and Rad6 was detected in 25% of the breast carcinomas. In vivo implantation of MCF10A-Rad6B cells produced hyperplastic lesions. These data reveal a potentially important role for transcriptionally active beta-catenin in the regulation of Rad6B gene expression, and link aberrant beta-catenin signaling with transcriptional deregulation of Rad6B and breast cancer development.

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

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 G(2)/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.

Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation.

We have isolated by differential RNA display a cDNA that is up-regulated in metastatic mammary tumor lines. This cDNA corresponds to HR6B, the yeast homologue of Rad6, a ubiquitin-conjugating enzyme, and a key player in postreplication repair and induced mutagenesis in the yeast. We show that Rad6 protein expressed in metastatic tumor lines is wild type and functional, because it is able to catalyze the transfer of ubiquitin to histone H2b and is predominantly localized in the nucleus as compared with cytoplasmic localization in normal or nonmetastatic mammary cells. This pattern of Rad6 protein expression/localization is not restricted to breast cancer cell lines, because human breast carcinomas display similar patterns of Rad6 up-regulation and nuclear localization suggesting that deregulation in expression of Rad6 may be an important step in transformation to malignant phenotype. Constitutive overexpression of exogenous human HR6B cDNA into normal-behaving MCF10A human breast epithelial cells induced cell-cell fusion that resulted in generation of multinucleated cells, centrosome amplification, multipolar mitotic spindles, aneuploidy, and ability for anchorage-independent growth. Double immunofluorescence labeling experiments demonstrated the colocalization of Rad6 protein with gamma-tubulin on centrosomes. This physical association of Rad6 with centrosomes is maintained throughout the interphase and mitotic phases of the cell cycle. The Rad6 protein exhibits notable alterations in distribution during interphase and mitotic stages of the cell cycle that are compatible with its function as a transcription factor. These findings suggest that Rad6 is an important ubiquitin-conjugating enzyme that may play a significant role in the maintenance of genomic integrity of mammalian cells and that an imbalance in the levels and activity of Rad6 could lead to chromosomal instability and transformation in vitro.

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 cell's 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.

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.

Functional analysis of MKP-1 and MKP-2 in breast cancer tamoxifen sensitivity.

Increased activation of ERK signaling has been reported in breast cancer models of acquired tamoxifen resistance. Here, we examined the expression of Mitogen-Activated Protein Kinase Phosphatases (MKPs) 1 and 2 following tamoxifen treatment and the effects of MKP-1/MKP-2 overexpression on tamoxifen sensitivity. Treatment of MCF7 breast cancer cells with tamoxifen increased MKP-2, but not MKP-1, protein levels. Overexpression of MKP-1 or MKP-2 inhibited estrogen-induced MCF7 cell proliferation compared to vector controls. MCF7-MKP-2 cells displayed significantly increased sensitivity to tamoxifen as compared to vector control or MCF7-MKP-1 cells. MKP-1 or MKP-2 overexpression eliminated ERK1/2 phosphorylation, suggesting that decreases in estrogen-induced proliferation of MKP-1 and MKP-2 overexpressing cells are due to ERK1/2 dephosphorylation. JNK1/2 activation was not detectable in any of these cells. These data suggest that tamoxifen-induced death of these cells is not dependent upon JNK signaling, but rather that ERK is the major MAPK driving their proliferation. MCF7-TAMR cells express higher levels of MKP-2 mRNA and protein than MCF7 cells. MKP-2 and phospho-ERK1/2 proteins are constitutively expressed in MCF7-TAMR cells, and activated JNK1/2 is not detectable. These data suggest that MKP-2 rather than MKP-1 is tamoxifen-regulated and that the elevated expression of MKP-2 in MCF7-TAMR cells potentially functions to restore tamoxifen sensitivity.

Melanoma Development and Progression Are Associated with Rad6 Upregulation and ß -Catenin Relocation to the Cell Membrane.

We have previously demonstrated that Rad6 and ß -catenin enhance each other's expression through a positive feedback loop to promote breast cancer development/progression. While ß -catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and ß -catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti- ß -catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. ß -Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear ß -catenin. ß -Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic ß -catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited ß -catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of ß -catenin and that ß -catenin and Rad6 play independent roles in melanoma development.

Rad6 is a Potential Early Marker of Melanoma Development.

Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/ß-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of ß-catenin stability in breast cancer cells. Similar to breast cancer, ß-catenin-activating mutations are rare in melanomas, and since ß-catenin signaling is implicated in melanoma, we examined the relationship between ß-catenin levels/activity and expression of ß-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, ß-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight ß-catenin protein levels and ß-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P=.0029). In contrast to strong ß-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development.

Comedo-DCIS is a precursor lesion for basal-like breast carcinoma: identification of a novel p63/Her2/neu expressing subgroup.

Basal breast cancer comprises ~15% of invasive ductal breast cancers, and presents as high-grade lesions with aggressive clinical behavior. Basal breast carcinomas express p63 and cytokeratin 5 (CK5) antigens characteristic of the myoepithelial lineage, and typically lack Her2/neu and hormone receptor expression. However, there is limited data about the precursor lesions from which they emerge. Here we wished to determine whether comedo-ductal carcinoma in situ (comedo- DCIS), a high-risk in situ breast lesion, serve as precursors for basal-like breast cancer. To determine this link, p63, CK5, Her2/neu, epidermal growth factor receptor (EGFR), estrogen receptor (ER) and progesterone receptor (PgR) expression were analyzed by immunohistochemistry in 17 clinical comedo- and 12 noncomedo-DCIS cases, and in tumors derived from unfractionated and CK5-overexpressing subpopulation (MCF10DCIS.com-CK5(high)) of MCF10DCIS.com cells, a model representative of clinical comedo-DCIS. p63 and Her2/neu coexpression was analyzed by immunofluorescence double labeling. A novel p63/CK5/Her2/neu expressing subpopulation of cells that are ER-/PgR-/EGFR- were identified in the myoepithelial and luminal areas of clinical comedo-DCIS and tumors derived from unfractionated MCF10DCIS.com and MCF10DCIS.com-CK5(high) cells. These data suggest that p63 and Her2/neu expressors may share a common precursor intermediate. P63, but not Her2/neu, expression was significantly associated (P = 0.038) with microinvasion/recurrence of clinical comedo-DCIS, and simultaneous expression of p63 and Her2/neu was marginally associated (P = 0.067) with comedo-DCIS. These data suggest that p63/Her2/neu expressing precursor intermediate in comedo-DCIS may provide a cellular basis for emergence of p63+/Her2/neu- or p63+/Her2/neu+ basal-like breast cancer, and that p63/Her2/neu coexpression may serve as biomarkers for identification of this subgroup of basal-like breast cancers.

Ocimum gratissimum retards breast cancer growth and progression and is a natural inhibitor of matrix metalloproteases.

Ocimum genus (a.k.a holy basil or tulsi) is a dietary herb used for its multiple beneficial pharmacologic properties including anti-cancer activity. Here we show that crude extract of Ocimum gratissimum (OG) and its hydrophobic and hydrophilic fractions (HB and HL) differentially inhibit breast cancer cell chemotaxis and chemoinvasion in vitro and retard tumor growth and temporal progression of MCF10ADCIS.com xenografts, a model of human breast comedo-ductal carcinoma in situ (comedo-DCIS). OG-induced inhibition of tumor growth was associated with decreases in basement membrane disintegration, angiogenesis and MMP-2 and MMP-9 activities as confirmed by in situ gelatin zymography and cleavage of galectin-3. There was also decrease in MMP-2 and MMP-9 activities in the conditioned media of OG-treated MCF10AT1 and MCF10AT1-EIII8 premalignant human breast cancer cells as compared with control. The MMP-2 and MMP-9 inhibitory activities of OG were verified in vitro using gelatin, a synthetic fluorogenic peptide and recombinant galectin-3 as MMP substrates. Mice fed on OG-supplemented drinking water showed no adverse effects compared with control. These data suggest that OG is non-toxic and that the anti-cancer therapeutic activity of OG may in part be contributed by its MMP inhibitory activity.