Christina Leah B. Kline

Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NFκB and microRNA network

BackgroundDiminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted.ResultsColon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NFκB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified ~700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NFκB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NFκB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity.ConclusionOur findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

The Akt Inhibitor ISC-4 Activates Prostate Apoptosis Response Protein-4 and Reduces Colon Tumor Growth in a Nude Mouse Model

Purpose: Prostate apoptosis response protein-4 (Par-4) sensitizes cells to chemotherapy; however, Akt1 inactivates Par-4. Previously we showed that Par-4–overexpressing colon cancer cells responded more readily to 5-fluorouracil (5-FU) than their wild-type counterparts. In this study we investigated (i) the effects of the Akt inhibitor, phenylbutyl isoselenocyanate (ISC-4), on tumor growth in nude mice and (ii) bystander effect of Par-4–overexpressing cells on wild-type tumor growth. Experimental Design: Mice (n = 80) were injected with wild-type HT29 human colon cancer cells in the right flank. Forty of the mice were also injected in the left flank with HT29 cells engineered to overexpress Par-4. The mice were treated with 5-FU, ISC-4, a combination, or vehicle. Results: ISC-4 reduced tumor growth, with or without 5-FU. When Par-4–overexpressing tumors were present, wild-type tumors grew more slowly compared to when no Par-4–overexpressing tumors were present. The level of Par-4 protein as well as the Par-4 binding protein, GRP78, was increased in wild-type cells growing in the same mouse as Par-4–overexpressing tumors compared with wild-type tumors growing without Par-4–overexpressing tumors. Conclusions: Par-4–overexpressing tumors exhibited a bystander effect on wild-type tumors growing distally in the same mouse. This suggests that gene therapy need not achieve total penetration to have a positive effect on tumor treatment. Inhibition of Akt with ISC-4 inhibited tumor growth and had a greater effect on cells overexpressing Par-4. The data indicate ISC-4 alone or in combination with Par-4 can greatly reduce tumor growth. Clin Cancer Res; 17(13); 4474–83. ©2011 AACR.

Delivery of PAR-4 plasmid in vivo via nanoliposomes sensitizes colon tumor cells subcutaneously implanted into nude mice to 5-FU

The prostate apoptosis response protein 4 (Par-4), a tumor suppressor, has been shown to induce apoptosis in cancer cells. While reduced Par-4 expression has been linked to survival of some cancers, its involvement in colon cancer has not been well documented. To explore the feasibility of increasing Par-4 in colon cancer to induce apoptosis, the human colon cancer cell line, HT29, was transfected to overexpress Par-4. In these cells, overexpressed Par-4 led to increased apoptosis in the presence of 5-fluorouracil. Subsequently, Par-4 cDNA was packaged in nanoliposomal particles. Treating cells with the Par-4 nanoliposomes also increased susceptibility to 5-FU. These nanoliposomes were used to deliver Par-4 plasmid to tumors growing in nude mice from wild type HT29 cells. Results showed that nanoliposomes effectively delivered plasmid DNA to tumors in vivo. Again, tumors in mice treated with the Par-4 nanoliposomes were more susceptible to 5-FU treatment. This suggests that upregulation of Par-4 expression is a potentially useful mechanism to enhance the current chemotherapeutic regimen for colon cancer. Packaging Par-4 cDNA in nanoliposomal particles is a promising delivery method to increase response to chemotherapy.

Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.

Preliminary observations indicate variable patterns of plasma 5-fluorouracil (5-FU) levels during dose optimization of infusional 5-FU in colorectal cancer patients

Efforts to improve efficacy and minimize toxicity have led to pharmacokinetic monitoring of plasma 5-Fluorouracil (5-FU) levels in colorectal cancer patients undergoing chemotherapy. We observed variation in basal 5-FU levels in 21 patients and significant variation during subsequent dose optimization. Tumor KRAS, BRAF mutations and TS mRNA levels were determined. Regimens included FOLFOX6+Avastin (N=8), FOLFOX6 (N=11), FOLFIRI (N=1) and FOLFOX4 (N=1). Mutations identified in tumors included G12V KRAS (N=2), G12A KRAS (N=1), and V600E BRAF (N=3). Six-of-eleven patients with normalized tumor TS mRNA levels 4.0 had a plasma 5-FU AUC of less than or equal to 20 mg.h/L. Approximately 2/3 of patients achieved therapeutic 5-FU AUC levels with 0-2 dose adjustments while a sub-group of ~1/3 of patients slowly achieved therapeutic levels (> 3-4 dose increases leading to supra-therapeutic 5-FU and subsequent reductions to lesser than original doses). Liver metastases and tumor TS levels did not fully account for variable 5-FU AUC optimization patterns. The 5-FU level during continuous infusion was half-therapeutic in one patient who received FOLFOX4. The observed heterogeneous patterns at baseline and during dose optimization of 5-FU levels suggest variations in 5-FU metabolism among treated patients. Physiological and/or genetic differences underlying heterogeneity in 5-FU levels during dose optimization require further study of patient demographics, single nucleotide polymorphisms in Dihydropyrimidine Dehydrogenase (DPD), TS, or other genes that impact 5-FU metabolism and gene expression changes in liver after 5-FU therapy.

Src kinase induces tumor formation in the c‐SRC C57BL/6 mouse

Src kinase has been linked as a causative agent in the progression of a number of cancers including colon, breast, lung and melanoma. Src protein and activity levels are increased in colorectal cancer and liver metastases arising secondary to colon cancer. However, although Src protein is increased in colon cancer as early as the adenomatous polyp stage, a role for Src in carcinogenesis has not been established. We developed the c‐SRC transgenic mouse in the C57BL/6 strain to address the issue of carcinogenesis in cells with high levels of Src expression. The transgene was constructed with the human c‐SRC gene downstream of the mouse metallothionein promoter to create zinc inducible gene expression. In these C57BL/6 mice, Src protein was increased in a number of tissues both with and without zinc induction. No additional carcinogenic agent was administered. After 20 months, mice were assessed for tumor development in the liver and GI tract, as well as other organs. Of the mice with the transgene, 15% developed tumors in the liver while no tumors were detected in wild type C57BL/6 mice. A further study was conducted by crossing c‐SRC C57BL/6 mice with p21 nullizygous mice to determine the effect of oncogene expression combined with inactivation of the tumor suppressor gene, p21. Addition of the c‐SRC transgene to the p21−/− background increased tumor formation almost 3‐fold, while it increased metastasis 6‐fold. The data from our study show, for the first time, that Src kinase may play a role in carcinogenesis.

Personalizing Colon Cancer Therapeutics: Targeting Old and New Mechanisms of Action

The use of pharmaceuticals for colon cancer treatment has been increasingly personalized, in part due to the development of new molecular tools. In this review, we discuss the old and new colon cancer chemotherapeutics, and the parameters that have been shown to be predictive of efficacy and safety of these chemotherapeutics. In addition, we discuss how alternate pharmaceuticals have been developed in light of a potential lack of response or resistance to a particular chemotherapeutic.

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU.

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.

Src activity alters α3 integrin expression in colon tumor cells

Src kinase has been linked to increased motility in the progression and metastasis of human colon cancer, although the mechanisms are not fully understood. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. This study examines the link between Src and integrin activity in the metastatic process in colon cancer cells. To determine Src involvement in integrin expression, the human colon cancer cell line, HCT116, was transfected with an activated Src construct and assayed for its ability to attach to and migrate across collagen and laminin. These cells attached more readily and migrated less rapidly on the extracellular matrix (ECM) than did cells transfected with empty vector. Examination of integrin levels showed a decrease in the α3 subunit in Src transfected cells as well as decreased cell surface localization of α3 integrin. The downregulation of α3 integrin was reversed by inhibition of Src and by inhibition of MAP kinase. Inhibition of α3 integrin using shRNA resulted in decreased MMP7 secretion, a possible cause of decreased invasion with low α3 integrin expression. This study shows that Src overexpression downregulates α3 integrin total protein expression and localization to the cell surface of HCT116 colon cancer cells. This indicates that Src activity may enhance metastasis by altering α3 integrin expression.

ONC201 demonstrates antitumor effects in both triple-negative and non–triple-negative breast cancers through TRAIL-dependent and TRAIL-independent mechanisms

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G1 phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290–8. ©2017 AACR.