Expansion of the 4-(Diethylamino)benzaldehyde Scaffold to Explore the Impact on Aldehyde Dehydrogenase Activity and Antiproliferative Activity in Prostate Cancer.

Aldehyde dehydrogenases (ALDHs) are overexpressed in various tumor types including prostate cancer and considered a potential target for therapeutic intervention. 4-(Diethylamino)benzaldehyde (DEAB) has been extensively reported as a pan-inhibitor of ALDH isoforms, and here, we report on the synthesis, ALDH isoform selectivity, and cellular potencies in prostate cancer cells of 40 DEAB analogues; three analogues (14, 15, and 16) showed potent inhibitory activity against ALDH1A3, and two analogues (18 and 19) showed potent inhibitory activity against ALDH3A1. Significantly, 16 analogues displayed increased cytotoxicity (IC50 = 10-200 µM) compared with DEAB (>200 µM) against three different prostate cancer cell lines. Analogues 14 and 18 were more potent than DEAB against patient-derived primary prostate tumor epithelial cells, as single agents or in combination treatment with docetaxel. In conclusion, our study supports the use of DEAB as an ALDH inhibitor but also reveals closely related analogues with increased selectivity and potency.

Intratumoural Cytochrome P450 Expression in Breast Cancer: Impact on Standard of Care Treatment and New Efforts to Develop Tumour-Selective Therapies.

Despite significant advances in treatment strategies over the past decade, selective treatment of breast cancer with limited side-effects still remains a great challenge. The cytochrome P450 (CYP) family of enzymes contribute to cancer cell proliferation, cell signaling and drug metabolism with implications for treatment outcomes. A clearer understanding of CYP expression is important in the pathogenesis of breast cancer as several isoforms play critical roles in metabolising steroid hormones and xenobiotics that contribute to the genesis of breast cancer. The purpose of this review is to provide an update on how the presence of CYPs impacts on standard of care (SoC) drugs used to treat breast cancer as well as discuss opportunities to exploit CYP expression for therapeutic intervention. Finally, we provide our thoughts on future work in CYP research with the aim of supporting ongoing efforts to develop drugs with improved therapeutic index for patient benefit.

The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary.

PURPOSE: Signaling by cancer stem cells (CSCs) is known to occur at least in part through conserved developmental pathways. Here, the role of one of these pathways, i.e., the hedgehog pathway, was evaluated in high-grade serous ovarian carcinoma (HGSOC). METHODS AND RESULTS: We found that in HGSOC, hedgehog inhibitors (HHIs) GANT61, LDE225 and GDC0449 reduced or inhibited the formation of spheroids enriched in CSCs. Primary malignant cells (PMCs) in ascites from HGSOC patients cultured in the presence of HHIs showed significant reduction in CSCs. Sonic hedgehog (SHH) significantly increased the expression of ALDH1A1, which was inhibited by GANT61. In the presence of a SHH neutralizing antibody (5E1), a significant reduction in the number of spheroids was observed in HGSOC-derived cell lines. Further, the motility, migration and clonogenic growth of the cells were significantly reduced by HHIs. In the presence of GANT61, a reduction of cells from PMCs in the G0 phase of the cell cycle was observed. The magnitude of difference in expression of Gli1 in tumors from the same HGSOC patients at presentation and at interval debulking surgery was greater in patients who had a recurrence on follow up. GANT61 also significantly inhibited the growth of CSCs in nude mice. Finally, RNA sequencing of HGSOC cells treated with GANT61 showed a significantly reduced expression of CSC markers. CONCLUSIONS: Our results indicate that the hedgehog pathway plays an important role in maintaining the integrity of CSCs in HGSOC and could be a potential therapeutic target.

ALDH1A1+ ovarian cancer stem cells co-expressing surface markers CD24, EPHA1 and CD9 form tumours in vivo.

One of the reasons for recurrence following treatment of high grade serous ovarian carcinoma (HGSOC) is the persistence of residual cancer stem cells (CSCs). There has been variability between laboratories in the identification of CSC markers for HGSOC. We have identified new surface markers (CD24, CD9 and EPHA1) in addition to those previously known (CD44, CD117 and CD133) using a bioinformatics approach. The expression of these surface markers was evaluated in ovarian cancer cell lines, primary malignant cells (PMCs), normal ovary and HGSOC. There was no preferential expression of any of the markers or a combination. All the markers were expressed at variable levels in ovarian cancer cell lines and PMCs. Only CD117 and CD9 were expressed in the normal ovarian surface epithelium and fallopian tube. Both ALDEFLUOR (ALDH1A1) and side population assays identified a small proportion of cells (<3%) separately that did not overlap with little variability in cell lines and PMCs. All surface markers were co-expressed in ALDH1A1+ cells without preference for one combination. The cell cycle analysis of ALDH1A1+ cells alone revealed that majority of them reside in G0/G1 phase of cell cycle. Further separation of G0 and G1 phases showed that ALDH1A1+ cells reside in G1 phase of the cell cycle. Xenograft assays showed that the combinations of ALDH1A1 + cells co-expressing CD9, CD24 or EPHA1 were more tumorigenic and aggressive with respect to ALDH1A1-cells. These data suggest that a combined approach could be more useful in identifying CSCs in HGSOC.

Expression of cancer stem cell markers CD24, EPHA1 and CD9 and their correlation with clinical outcome in epithelial ovarian tumours.

BACKGROUND: There has been variability between laboratories in the identification of cancer stem cells (CSCs) markers for epithelial ovarian cancer (EOC). We have evaluated three new surface markers for EOC to identify CSCs precisely. METHODS: Three new putative CSCs specific surface markers CD9, CD24 and EPHA1 identified by a bioinformatics approach were evaluated in normal ovary, fallopian tube and ovarian tumours. RESULTS: The expression of CD9 alone was observed in normal ovarian surface epithelium and fallopian tube whereas CD24 and EPHA1 were not expressed (n= 5). CD24 was expressed in all tumours (N= 101) while CD9 and EPHA1 were expressed in 89 and 71 tumours, respectively. The statistical analysis showed significant correlation of the stage of the disease (p< 0.0001), type of surgery (p< 0.0001) and residual disease (p< 0.0001) with overall survival. Although expression of CD9, CD24 and EPHA1 was observed in the majority of tumours there was no significant correlation with outcome. In patients who underwent primary surgery, increased expression of CD24 significantly correlated with poor survival. The expression of CD24 was significantly reduced (p< 0.002) upon analysis of paired sections from patients prior to surgery and at interval debulking surgery (n= 16). CONCLUSION: These findings suggest that overexpression of these new markers may be useful in identifying and targeting ovarian CSCs and CD24 may be a putative CSCs marker in ovarian cancer.

Analysis of Human Stem Cell Transcription Factors.

Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in both human embryonic stem cells (hESCs) and cancer stem cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency. This article evaluates the expression of variants of the main stem cell-specific transcription factors NANOG and OCT4 critically and accurately with specific primers designed for identifying the most important variants that maintain stemness. We have examined four variants of NANOG along with a processed pseudogene and seven variants of OCT4 in human teratocarcinoma cell lines (NTERA2D1, SuSa, GCT-27, and 833KE), hESCs, and ovarian cancer cells by reverse transcriptase-polymerase chain reaction. In addition, we have examined their expression in NTERA2D1 cells on differentiation with all-trans-retinoic-acid. We show that NANOG1 is expressed in all teratocarcinoma cells and can be distinguished from NANOGP8, which is an expressed pseudogene. NANOG2 was not expressed in any of the cell lines, including ESCs. OCT4A was expressed in all cells, whereas the variant OCT4B-variant 3 was expressed only in NTERA2D1 cells. On differentiation of NTERA2D1 with retinoic acid, only NANOGP8 and OCT4A were expressed. In ovarian cancer cells, only 3/6 expressed NANOG1 and OCT4A. All malignant cells from patients with ovarian cancer (N = 6) expressed NANOG1 and OCT4A. These results demonstrate the necessity to precisely evaluate the expression of stem cell transcription factors when defining stemness.

Cancer stem cells contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary.

The origin of blood and lymphatic vessels in high-grade serous adenocarcinoma of ovary (HGSOC) is uncertain. We evaluated the potential of cancer stem cells (CSCs) in HGSOC to contribute to their formation. Using spheroids as an in vitro model for CSCs, we have evaluated their role in primary malignant cells (PMCs) in ascites from previously untreated patients with HGSOC and cell lines. Spheroids from PMCs grown under specific conditions showed significantly higher expression of endothelial, pericyte and lymphatic endothelial markers. These endothelial and lymphatic cells formed tube-like structures, showed uptake of Dil-ac-LDL and expressed endothelial nitric oxide synthase confirming their endothelial phenotype. Electron microscopy demonstrated classical Weibel-Palade bodies in differentiated cells. Genetically, CSCs and the differentiated cells had a similar identity. Lineage tracking using green fluorescent protein transfected cancer cells in nude mice confirmed that spheroids grown in stem cell conditions can give rise to all three cells. Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor inhibited the differentiation of spheroids to endothelial cells in vitro. These results suggest that CSCs contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary, which can be inhibited.

Therapeutic antibodies against cancer stem cells: a promising approach.

Monoclonal antibodies have been extensively used to treat malignancy along with routine chemotherapeutic drugs. Chemotherapy for metastatic cancer has not been successful in securing long-term remission of disease. This is in part due to the resistance of cancer cells to drugs. One aspect of the drug resistance is the inability of conventional drugs to eliminate cancer stem cells (CSCs) which often constitute less than 1-2% of the whole tumor. In some tumor types, it is possible to identify these cells using surface markers. Monoclonal antibodies targeting these CSCs are an attractive option for a new therapeutic approach. Although administering antibodies has not been effective, when combined with chemotherapy they have proved synergistic. This review highlights the potential of improving treatment efficacy using functional antibodies against CSCs, which could be combined with chemotherapy in the future.

Cancer Stem Cells - Are Surface Markers Alone Sufficient?

The identification of Cancer Stem Cells (CSCs) in leukemia has opened a new field in cancer research. This has led to the identification of similar cells in other types of cancer. CSCs express distinct surface markers and functional properties which distinguish them from the rest of the cells within a tumor. Due to variability in identification of CSCs in a particular type of cancer (except brain, breast and leukemia), surface markers alone may not be sufficient. It is critical to identify and isolate this small population of cells from the heterogeneous tumors to understand their pathogenesis. Identification of surface markers together with intrinsic properties of CSCs like colony formation, Hoechst exclusion or ALDEFLUOR assay may be useful in isolating more primitive and highly pure CSCs from a heterogeneous population of malignant cells. This review critically analyses various techniques and methods along with their advantages and disadvantages that are employed in identifying CSCs from different types of cancers.