The anti-melanoma activity of dinaciclib, a cyclin-dependent kinase inhibitor, is dependent on p53 signaling.

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.

Interleukin-1ß promotes skeletal colonization and progression of metastatic prostate cancer cells with neuroendocrine features.

Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1ß (IL-1ß), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1ß by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1ß in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1ß secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1ß and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1ß supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.

Bortezomib induces apoptosis in esophageal squamous cell carcinoma cells through activation of the p38 mitogen-activated protein kinase pathway.

Esophageal squamous cell carcinoma (ESCC) is an exceptionally drug-resistant tumor with a 5-year survival rate <5%. From an initial drug screen, we identified bortezomib as having robust activity in ESCC lines. Mechanistically, bortezomib induced a G2-M-phase cell cycle arrest and p53-independent apoptosis associated with caspase cleavage and Noxa induction. Bortezomib also showed excellent activity in organotypic culture and in vivo models of ESCC. Biochemically, bortezomib treatment activated the p38 and c-Jun NH2-termnial kinase stress-activated mitogen-activated protein kinase (MAPK) pathways and induced phospho-H2AX activity. Although H2AX is known to cooperate with c-Jun NH2-termnial kinase to induce apoptosis following UV irradiation, knockdown of H2AX did not abrogate bortezomib-induced apoptosis. Instead, blockade of p38 MAPK signaling, using either small interfering RNA or a pharmacologic inhibitor, reversed bortezomib-induced apoptosis and the up-regulation of Noxa. Radiation therapy is known to activate the p38 MAPK pathway and is a mainstay of ESCC treatment strategies. In a final series of studies, we showed that the coadministration of bortezomib with irradiation led to enhanced p38 MAPK activity and a significant reduction in colony formation. We therefore suggest that p38 MAPK pathway activation is an excellent potential therapeutic strategy in ESCC. It is further suggested that bortezomib could be added to existing ESCC therapeutic regimens.

Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas.

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E-mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E-mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.

The essential role of fibroblasts in esophageal squamous cell carcinoma-induced angiogenesis.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is known to be a highly angiogenic tumor. Here, we investigated the role of the stromal fibroblasts in the ESCC-induced angiogenic response using a novel 3-dimensional model. METHODS: A novel assay was developed where cocultures of ESCC and esophageal fibroblasts induced human microvascular endothelial cell (HMVEC) vascular network formation in a 3-dimensional collagen gel. Biochemical studies showed that the ESCC-induced activation of the fibroblasts was required to induce vascular network formation via a transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF)-dependent pathway. RESULTS: Conditioned media from a panel of 4 ESCC lines transdifferentiated normal esophageal fibroblasts into myofibroblasts via TGF-beta signaling. The presence of fibroblasts was essential for efficient HMVEC network formation, and the addition of ESCC cells to these cultures greatly enhanced the angiogenic process. The role of TGF-beta in this process was shown by the complete inhibition of network formation following TGF-beta inhibitor treatment. Finally, we showed that ESCC-derived TGF-beta regulates angiogenesis through the release of VEGF from the fibroblasts and that the VEGF release was blocked following TGF-beta inhibition. CONCLUSIONS: This study shows the essential role of fibroblasts in the ESCC angiogenic-induced response and suggests that the pharmacologic targeting of the TGF-beta signaling axis could be of therapeutic benefit in this deadly disease.

In vitro three-dimensional tumor microenvironment models for anticancer drug discovery.

Anticancer drug discovery has long been hampered by the poor predictivity of the preclinical models. There is a growing realization that the tumor microenvironment is a critical determinant of the response of cancer cells to therapeutic agents. The past 5 years have seen a great deal of progress in our understanding of how the three-dimensional microenvironment modulates the signaling behavior of tumor cells. The present review discusses how three-dimensional in vitro cell culture models can benefit cancer drug discovery through an accurate modeling of the tumor microenvironment, leading to more physiologically relevant experimental outcomes.

Dysregulation of claudin-7 leads to loss of E-cadherin expression and the increased invasion of esophageal squamous cell carcinoma cells.

The claudins constitute a 24-member family of proteins that are critical for the function and formation of tight junctions. Here, we examine the expression of claudin-7 in squamous cell carcinoma (SCC) of the esophagus and its possible role in tumor progression. In the normal esophagus, expression of claudin-7 was confined to the cell membrane of differentiated keratinocytes. However, in the tumor samples, claudin-7 expression is often lost or localized to the cytoplasm. Assaying esophageal SCC lines revealed variable expression of claudin-7, with some lacking expression completely. Knockdown of claudin-7 in SCC cell lines using a small interfering RNA approach led to decreased E-cadherin expression, increased cell growth, and enhanced invasion into a three-dimensional matrix. The opposite was observed when claudin-7 was overexpressed in esophageal SCC cells lacking both claudin-7 and E-cadherin. In this context, the claudin-7-overexpressing cells became more adhesive and less invasive associated with increased E-cadherin expression. In summary, we demonstrate that claudin-7 is mislocalized during the malignant transformation of esophageal keratinocytes. We also demonstrate a critical role for claudin-7 expression in the regulation of E-cadherin in these cells, suggesting this may be one mechanism for the loss of epithelial architecture and invasion observed in esophageal SCC.

Life isn't flat: taking cancer biology to the next dimension.

Classically, most cell culture experiments have been performed under adherent 2D conditions. Cells in the human body grow within an organized 3D matrix, surrounded by other cells. The behavior of individual cells is controlled through their interactions with their immediate neighbors and the extracellular matrix. The complex summation of these multiple signals determines whether a given cell undergoes differentiation, apoptosis, proliferation, or invasion. In 2D culture many of these complex interactions are lost. As a result, there are a growing number of studies which report differences in phenotype, cellular signaling, cell migration, and drug responses when the same cells are grown under 2D or 3D culture conditions. One potential application of these techniques is to anticancer drug discovery, which has long been hampered by the lack of good preclinical models. Compounds with good antitumor activity in 2D cell culture models often fail to translate into the clinic. Here we suggest that the response of cancer cells to drugs is determined in part by the 3D tumor microenvironment and discuss models to re-create the 3D tumor microenvironment in vitro. It is likely that the adoption of these and other 3D models will allow us to more closely re-create the behavior of the tumor in vivo which may lead to identifying better anticancer drug candidates at an earlier stage of development.

Primary amelanotic melanoma of the esophagus.

Primary melanoma of the esophagus (PME) is an uncommon malignancy with less than 250 cases reported in the literature. Amelanotic PME is exceedingly rare and accounts for 10-25% of melanomas of the esophagus. A 59-year-old male with a history of mild dysphagia, heartburn, moderate anorexia and weight loss for 1 month is described. Barium swallow examination and videogastroscopy showed a polypoid, ulcerated mass located 30-38 cm from the incisors. No skin or eye melanoma lesions were found. Five biopsy samples were obtained. Histological examinations revealed proliferation of large, loosely cohesive cells of variable shapes and prominent central nucleoli in the deep mucosa. Immunohistochemical findings included positive vimentin, protein S-100, Melan A, and HMB-45, and negative AE1/AE3, CD 17, and desmin. A total transhiatal esophagectomy with high cervical esophagogastric anastomosis was performed. Peritumoral lymph nodes revealed malignant invasion. A diagnosis of primary amelanotic melanoma of the esophagus was made. Fourteen months after diagnosis the patient developed disseminated PME.

Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases.

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions.

Targeting the stromal fibroblasts: a novel approach to melanoma therapy.

Classically, cancer is thought of as a genetic disease, where the step-wise acquisition of mutations initiates and drives progression. More recent thinking posits that, although cancers are initiated through genetic mutation, progression is often the result of dynamic interactions between the tumor cells and their surrounding environment.