The small GTP-binding protein Rac is a downstream e ector of the oncogene product p21-ras. Rac is involved in actin polymerization, Jun kinase activation, and intracellular superoxide anion production, through distinct pathways in tumor cells. Here we investigated the role of activated Rac in the response of tumor cells to apoptosis triggered by anti-cancer drugs or the cell surface death receptor CD95. Using M14 melanoma cells stably transfected with a constitutively active form of Rac1, we show that activated Rac inhibits tumor cell response to apoptosis. The inhibitory e ect of activated Rac on apoptotic signaling is mediated by the interaction of Rac with intracellular oxidase and the subsequent production of superoxide, which is supported by experiments performed with M14 and NIH3T3 cells transiently transfected with the loss-of-function mutants of Rac in an activated RacV12 background. Consistent with these ®ndings, we also demonstrate that inhibition of the Rac pathway in the HaRas-expressing T24 bladder carcinoma cell line induces a decrease in superoxide anion concentration, and results in a signi®cant increase in tumor cell sensitivity to apoptosis. These ®ndings demonstrate the existence of a novel Racdependent survival pathway mediated by intracellular superoxide in tumor cells. Oncogene (2001) 20, 6263 ± 6268.

Tumors with BRCA germline mutations are defective in repairing DNA double-strand breaks (DSB) through homologous recombination (HR) pathways, making them sensitive to PARP inhibitors (PARPi). However, BRCA germline mutations are rare in prostate cancer limiting the ability to therapeutically target these pathways. This study investigates whether histone deacetylase (HDAC) inhibitors (HDACi), reported to modulate DSB repair pathways in sporadic cancers, can downregulate DSB repair pathways and sensitize prostate cancer cells to PARPi. Prostate cancer cells cotreated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) and the PARPi, olaparib, demonstrated a synergistic decrease in cell viability compared with single-agent treatment (combination index < 0.9), whereas normal prostatic cells did not. Similarly, clonogenicity was significantly decreased after cotreatment. Flow cytometric cell-cycle analysis and Annexin-V staining revealed significant apoptosis upon treatment with SAHAþolaparib. This coincided with increased DNA damage observed by immunofluorescence microscopy analysis of gH2AX foci, a marker of DSBs. In addition, immunoblot analysis showed a significant and persistent increase in nuclear gH2AX levels. Both SAHA and olaparib downregulated the expression of HR-related proteins, BRCA1 and RAD51, whereas SAHA þ olaparib had an additive effect on RAD51. Silencing RAD51 sensitized prostate cancer cells to SAHA and olaparib alone. Collectively, cotreatment with HDACi and PARPi downregulated HR-related protein expression and concomitantly increased DNA damage, resulting in prostate cancer cell death.Implications: These findings provide a strong rationale for supporting the use of combined HDAC and PARP inhibition in treating advanced prostate cancer.

BackgroundCirculating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge.MethodsCoherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line.ResultsOne hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells.ConclusionsIntracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.

Tumor-derived extracellular vesicles (EVs) are emerging as an important mode of intercellular communication, capable of transferring biologically active molecules that facilitate the malignant growth and metastatic process. CD133 (Prominin-1), a stem cell marker implicated in tumor initiation, differentiation and resistance to anti-cancer therapy, is reportedly associated with EVs in various types of cancer. However, little is known about the factors that regulate the release of these CD133 EVs. Here, we report that the HDAC6 inhibitor tubacin promoted the extracellular release of CD133 EVs from human FEMX-I metastatic melanoma and Caco-2 colorectal carcinoma cells, with a concomitant downregulation of intracellular CD133. This effect was specific for tubacin, as inhibition of HDAC6 deacetylase activity by another selective HDAC6 inhibitor, ACY-1215 or the pan-HDAC inhibitor trichostatin A (TSA), and knockdown of HDAC6 did not enhance the release of CD133 EVs. The tubacin-induced EV release was associated with changes in cellular lipid composition, loss of clonogenic capacity and decrease in the ability to form multicellular aggregates. These findings indicate a novel potential anti-tumor mechanism for tubacin in CD133-expressing malignancies. J. Cell. Biochem. 118: 4414-4424, 2017. © 2017 Wiley Periodicals, Inc.