Cancer cells with the surface marker profile CD44+/CD24- have previously been described to possess cancer stem cell-like properties. This manuscript evaluates those properties in ovarian cancer cell lines. The proportion of CD44+/CD24- cells corresponded to the clinical aggressiveness of each ovarian cancer cell line histologic subtype. CD44+/CD24- cells demonstrated enhanced progressive differentiation as well as showing a 60-fold increase in Matrigel invasion in both SKOV3 and OV90 cell lines (p < 0.001 each) compared to other phenotypes. CD44+/CD24- demonstrated significant resistance to all chemotherapy agents used in all cell lines, with a 71-93 % increase in resistance compared with baseline. Using a threshold of 25 % CD44+/CD24- ovarian cancer cells found in ascites, patients with >25 % CD44+/CD24- were significantly more likely to recur (83 vs. 14 %, p = 0.003) and had shorter median progression-free survival (6 vs. 18 months, p = 0.01). In conclusion, the CD44+/CD24- phenotype in ovarian cancer cells demonstrate cancer stem cell-like properties of enhanced differentiation, invasion, and resistance to chemotherapy. This CD44+/CD24- phenotype correlates to clinical endpoints with increased risk of recurrence and shorter progression-free survival in patients with ovarian cancer.

ObjectiveAldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.MethodsIsogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.ResultsALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.ConclusionThis data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

Extracellular high mobility group box 1 (HMGB1) is a novel cytokine that takes part in the processes of inflammation, tissue damage and regeneration. Mesenchymal stem cells (MSCs) are adult stem cells characterized by their inherently suppressive activities on inflammative and allo-immune reactions. In the present study, we have addressed whether HMGB1 could affect the biological properties of human bone marrow MSCs. Transwell experiments showed that HMGB1 induced MSC migration and this effect could not be hampered by a blocking antibody against the receptor for advanced glycation end products (RAGE). MSCs exposed to HMGB1 were negative for CD31, CD45, CD80, and HLA-DR, and displayed equal levels of CD73, CD166, and HLA-ABC compared with their counterparts, but HMGB1 profoundly suppressed MSC proliferation in a dose-dependent manner as evaluated by carboxyfluorescein diacetate succinmidyl ester dye dilution assay. Furthermore, HMGB1 triggered the differentiation of MSCs into osteoblasts as identified by histochemical staining, traditional RT-PCR and real-time RT-PCR analysis on mRNA expression of lineage-specific molecular markers. The differentiation-inductive activity could neither be inhibited by RAGE neutralizing antibody. Moreover, HMGB1-treated MSCs displayed unchanged suppressive activity on in vitro lymphocyte cell proliferation elicited by ConA. Collectively, the data suggest that MSCs are a target of HMGB1.

DNAJB6 also known as mammalian relative of DnaJ (MRJ) encodes a highly conserved member of the DnaJ/Hsp40 family of co-chaperone proteins that function with Hsp70 chaperones. DNAJB6 is widely expressed in all tissues, with higher expression levels detected in the brain. DNAJB6 is involved in diverse cellular functions ranging from murine placental development, reducing the formation and toxicity of mis-folded protein aggregates, to self-renewal of neural stem cells. Involvement of DNAJB6 is implicated in multiple pathologies such as Huntington's disease, Parkinson's diseases, limb-girdle muscular dystrophy, cardiomyocyte hypertrophy and cancer. This review summarizes the important involvement of the spliced isoforms of DNAJB6 in various pathologies with a specific focus on the emerging roles of human DNAJB6 in cancer and the underlying molecular mechanisms.

The hedgehog (Hh) pathway has been shown to be activated in numerous malignancies as well as in cancer stem cells. We sought to determine the importance of the Hh pathway in regulating growth and development of ovarian cancer spheroid-forming cells (SFCs). Ovarian cancer cell lines (ES2, TOV112D, OV90, and SKOV3) as well as a normal ovarian epithelial cell line (IOSE80) were grown in non-adherent growth conditions to form SFCs. Western blot analysis was used to determine the expression of Hh pathway proteins SMOH, PTCH, GLI1. SFCs were treated with Hh agonists (SHH and IHH) as well as an Hh inhibitor (cyclopamine) to determine changes in spheroid growth and survival. All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions while IOSE80 failed to form SFCs. Compared to IOSE80, ovarian cancer cell lines demonstrated significant activation of the Hh pathway as defined by increased expression of intranuclear GLI1. Both Hh agonists demonstrated significant increases in spheroid volume of at least 42-fold for SHH-treated cells and 46-fold for IHH-treated cells. With regard to survival, SFCs were 30-50% more resistant to cyclopamine than their corresponding monolayer cells. Despite this resistance, inhibition of the Hh pathway with cyclopamine prevented further growth of SFCs with a 10-, 5-, and 4-fold restriction in growth for ES2, SKOV3, and TOV112D, respectively. The hedgehog pathway appears to be important in regulating growth of ovarian cancer spheroid-forming cells. The activation and inhibition of this pathway demonstrates significant correlation to enhanced growth and growth restriction, respectively.

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Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

The expression of the tumor suppressor Merlin is compromised in nervous system malignancies due to genomic aberrations. We demonstrated for the first time, that in breast cancer, Merlin protein expression is lost due to proteasome-mediated elimination. Immunohistochemical analysis of tumor tissues from patients with metastatic breast cancer revealed characteristically reduced Merlin expression. Importantly, we identified a functional role for Merlin in impeding breast tumor xenograft growth and reducing invasive characteristics. We sought to determine a possible mechanism by which Merlin accomplishes this reduction in malignant activity. We observed that breast and pancreatic cancer cells with loss of Merlin show an aberrant increase in the activity of β-catenin concomitant with nuclear localization of β-catenin. We discovered that Merlin physically interacts with β-catenin, alters the sub-cellular localization of β-catenin, and significantly reduces the protein levels of β-catenin by targeting it for degradation through the upregulation of Axin1. Consequently, restoration of Merlin inhibited β-catenin-mediated transcriptional activity in breast and pancreatic cancer cells. We also present evidence that loss of Merlin sensitizes tumor cells to inhibition by compounds that target β-catenin-mediated activity. Thus, this study provides compelling evidence that Merlin reduces the malignant activity of pancreatic and breast cancer, in part by suppressing the Wnt/β-catenin pathway. Given the potent role of Wnt/β-catenin signaling in breast and pancreatic cancer and the flurry of activity to test β-catenin inhibitors in the clinic, our findings are opportune and provide evidence for Merlin in restraining aberrant activation of Wnt/β-catenin signaling.