Dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells upon drug-induced DNA damage under hypoxia.

Intratumoral hypoxia has been proposed to create a "mutator" phenotype through downregulation of DNA repair, leading to increased genomic instability and drug resistance. Such downregulation of DNA repair has been proposed to sensitize hypoxic cancer cells to DNA-damaging agents and inhibitors of DNA repair. Here, we showed that prostate cancer cells with mutant p53 were resistant to the poly(ADP-ribose) polymerase inhibitor, veliparib (2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide, dihydrochloride; Abbott Laboratories, Abbott Park, IL), and the DNA-damaging topoisomerase I inhibitor camptothecin-11 (CPT-11) or SN38 (7-ethyl-10-hydroxycamptothecin) under hypoxia. Upregulation of Rad51 by E2F1 upon DNA damage under hypoxia contributed to such resistance, which was reversed by either inhibiting RAD51 transcription with small interfering RNA or by expressing wild-type p53 in the p53 null prostate cancer line. Accumulation of endogenous p53 but not E2F1 and suppressed RAD51 transcription was observed in prostate cancer line with wild-type p53 after DNA damage under hypoxia. Combining veliparib with CPT-11 significantly enhanced DNA damage and apoptosis under both hypoxic and normoxic culture conditions. Such enhanced DNA damage and antitumor activities were seen in the presence of Rad51 upregulation and confirmed in vivo with PC3 mouse xenografts. These data illustrate a dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells' response to hypoxia and DNA damage. The veliparib and CPT-11 combination can be further explored as a treatment of metastatic castration-resistant prostate cancers that have frequent p53 mutations and enriched genomic instability.

The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex.

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterize a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicate that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex.

Erythropoietin supports the survival of prostate cancer, but not growth and bone metastasis.

Erythropoietin (Epo) is used in clinical settings to enhance hematopoietic function and to improve the quality of life for patients undergoing chemotherapy by reducing fatigue and the need for transfusions. However, several meta-analyses have revealed that Epo treatments are associated with an increased risk of mortality in cancer patients. In this study, we examined the role of Epo in prostate cancer (PCa) progression, using in vitro cell culture systems and in vivo bone metastatic assays. We found that Epo did not stimulate the proliferation of PCa cell lines, but did protect PCa cells from apoptosis. In animal models of PCa metastasis, no evidence was found to support the hypothesis that Epo enhances metastasis. Together, these findings suggest that Epo may be useful for treating severe anemia in PCa patients without increasing metastatic risk.

Role of transcriptional corepressor CtBP1 in prostate cancer progression.

Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferation and invasion. Expression profiling studies of prostate cancer cell lines revealed that multiple tumor suppressor genes are repressed by CtBP1. Furthermore, our studies indicate a role for CtBP1 in conferring radiation resistance to prostate cancer cell lines. In vivo studies using chicken chorioallantoic membrane assay, xenograft studies, and murine metastasis models suggested a role for CtBP1 in prostate tumor growth and metastasis. Taken together, our studies demonstrated that dysregulated expression of CtBP1 plays an important role in prostate cancer progression and may serve as a viable therapeutic target.

Prevalence of prostate cancer metastases after intravenous inoculation provides clues into the molecular basis of dormancy in the bone marrow microenvironment.

Bone is the preferred metastasis site of advanced prostate cancer (PCa). Using an in vivo murine model of human PCa cell metastasis to bone, we noted that the majority of animals that develop skeletal metastasis have either spinal lesions or lesions in the bones of the hindlimb. Much less frequently, lesions develop in the bones of the forelimb. We therefore speculated whether the environment of the forelimb bones is not permissive for the growth of PCa. Consequently, data on tumor prevalence were normalized to account for the number of PCa cells arriving after intravascular injection, marrow cellularity, and number of hematopoietic stem cell niches. None of these factors were able to account for the observed differences in tumor prevalence. An analysis of differential gene and protein levels identified that growth arrest specific-6 (GAS6) levels were significantly greater in the forelimb versus hindlimb bone marrow. When murine RM1 cells were implanted into subcutaneous spaces in immune competent animals, tumor growth in the GAS6(-/-) animals was greater than in GAS6(+/+) wild-type animals. In an osseous environment, the human PC3 cell line grew significantly better in vertebral body transplants (vossicles) derived from GAS6(-/-) animals than in vossicles derived from GAS6(+/+) animals. Together, these data suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy. Importantly, these data suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.

AT-101 (R-(-)-gossypol acetic acid) enhances the effectiveness of androgen deprivation therapy in the VCaP prostate cancer model.

Prostate cancer remains a leading cause of cancer death in American men. Androgen deprivation therapy (ADT) is the most common treatment for advanced prostate cancer patients; however, ADT fails in nearly all cases resulting in castration resistant or androgen-insensitive (AI) disease. In many cases, this progression results from dysregulation of the pro-survival Bcl-2 family proteins. Inhibition of pro-survival Bcl-2 family proteins, therefore, may be an effective strategy to delay the onset of AI disease. Gossypol, a small molecule inhibitor of pro-survival Bcl-2 family proteins, has been demonstrated to inhibit AI prostate cancer growth. The apoptotic effect of gossypol, however, has been demonstrated to be attenuated by the presence of androgen in a prostate cancer xenograft mouse model (Vertebral Cancer of Prostate [VCaP]) treated with AT-101 (R-(-)-gossypol acetic acid). This study was undertaken to better understand the in vitro effects of androgen receptor (AR) on AT-101-induced apoptosis. VCaP cells treated with AT-101 demonstrated an increase in apoptosis and downregulation of Bcl-2 pro-survival proteins. Upon AR activation in combination with AT-101 treatment, apoptosis is reduced, cell survival increases, and caspase activation is attenuated. Akt and X inhibitor of apoptosis (XIAP) are downregulated in the presence of AT-101, and AR stimulation rescues protein expression. Combination treatment of bicalutamide and AT-101 increases apoptosis by reducing the expression of these pro-survival proteins. These data suggest that combination therapy of AT-101 and ADT may further delay the onset of AI disease, resulting in prolonged progression-free survival of prostate cancer patients.

The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment.

CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3(lucCCL2)) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3(lucMock). In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3(lucCCL2) enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3(luc), which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.

Ecological therapy for cancer: defining tumors using an ecosystem paradigm suggests new opportunities for novel cancer treatments.

We propose that there is an opportunity to devise new cancer therapies based on the recognition that tumors have properties of ecological systems. Traditionally, localized treatment has targeted the cancer cells directly by removing them (surgery) or killing them (chemotherapy and radiation). These modes of therapy have not always been effective because many tumors recur after these therapies, either because not all of the cells are killed (local recurrence) or because the cancer cells had already escaped the primary tumor environment (distant recurrence). There has been an increasing recognition that the tumor microenvironment contains host noncancer cells in addition to cancer cells, interacting in a dynamic fashion over time. The cancer cells compete and/or cooperate with nontumor cells, and the cancer cells may compete and/or cooperate with each other. It has been demonstrated that these interactions can alter the genotype and phenotype of the host cells as well as the cancer cells. The interaction of these cancer and host cells to remodel the normal host organ microenvironment may best be conceptualized as an evolving ecosystem. In classic terms, an ecosystem describes the physical and biological components of an environment in relation to each other as a unit. Here, we review some properties of tumor microenvironments and ecological systems and indicate similarities between them. We propose that describing tumors as ecological systems defines new opportunities for novel cancer therapies and use the development of prostate cancer metastases as an example. We refer to this as "ecological therapy" for cancer.

In vivo evaluation of AT-101 (R-(-)-gossypol acetic acid) in androgen-independent growth of VCaP prostate cancer cells in combination with surgical castration.

PURPOSE: Upregulation of Bcl-2 family members is a well-established mechanism in the development of androgen-independent prostate cancer. Inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1 may delay the transition to androgen-independent growth. EXPERIMENTAL DESIGN: We have established a prostate cancer model with VCaP prostate cancer cells in vivo to study the transition to androgen independence. Here, we investigated the efficacy of AT-101 (R-(-)-gossypol acetic acid; a pan small molecule inhibitor of Bcl-2, Bcl-x(L), and Mcl-1) in combination with surgical castration to delay the onset of androgen-independent growth in vivo. RESULTS: AT-101 (15 mg/kg, per os (p.o.) 5 days/week) in combination with surgical castration delayed the onset of androgen-independent prostate cancer growth in vivo. In addition, we demonstrate the induction of caspase-9-and caspase-3-dependent induction of apoptosis following AT-101 treatment in vitro which was accompanied by an AT-101-induced downregulation of Bcl-2 and Mcl-1 mRNA and protein expression. CONCLUSIONS: We conclude that AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer in vivo by disrupting the antiapoptotic activity of Bcl-2 upregulation during the transition to androgen independence. Further studies are needed to define the mechanism of action by which AT-101 attenuates the expression of Bcl-2 and Mcl-1 and to characterize the potential for AT-101 in combination with hormone therapy.

The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage.

We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.