Kamila Wolanin

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.

Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells

Induction of senescence has been proposed as a possible in vivo tumor response to anticancer treatment. Senescent cancer cells are often polyploid, however, their route to polyploidy is poorly recognized (endoreduplication versus aberrant mitoses). We showed that after treatment of HCT116 cells with a low dose of doxorubicin most of them stopped proliferation as documented by SA-beta-galactosidase activity and the lack of Ki67 expression. Increased expression of other common senescence markers, p53, p21 and cyclin D1, was also observed. The cells became giant, polyploid and polymorphic, with multinucleated cells comprising a substantial fraction. The vast majority of the doxorubicin-treated cells did not enter mitoses, as evidenced by mitotic index analysis, as well as by the predominantly cytoplasmic localization of cyclin B1 and a lack of separation of multiplied centrosomes. This allowed us to conclude that doxorubicin-treated HCT116 cells underwent endoreduplication. However, the rare events of aberrant mitoses of polyploid cells observed by us led to aneuploid progeny as was documented by cytogenetic analysis of survivors. Thus, a senescence-inducing treatment of HCT116 cancer cells had a dual effect-it stopped the proliferation of the majority of the cells, but also led to the appearance of proliferating aneuploid ones.

Curcumin Affects Components of the Chromosomal Passenger Complex and Induces Mitotic Catastrophe in Apoptosis-Resistant Bcr-Abl-Expressing Cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G2-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells—morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia. (Mol Cancer Res 2006;4(7):457–69)

The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells

Activation of adaptive mechanisms plays a crucial role in cancer progression and drug resistance by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Our previous work showed that ER stress is induced in chronic myeloid leukemia (CML) cells. Herein, we demonstrate that the PERK-eIF2α phosphorylation pathway is upregulated in CML cell lines and CD34+ cells from CML patients and is associated with CML progression and imatinib resistance. We also show that induction of apoptosis by imatinib results in the downregulation of the PERK-eIF2α phosphorylation arm. Furthermore, we demonstrate that inactivation of the PERK-eIF2α phosphorylation arm decreases the clonogenic and proliferative capacities of CML cells and sensitizes them to death by imatinib. These findings provide evidence for a pro-survival role of PERK-eIF2α phosphorylation arm that contributes to CML progression and development of imatinib resistance. Thus, the PERK-eIF2α phosphorylation arm may represent a suitable target for therapeutic intervention for CML disease.

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

TOPBP1 acts in homologous recombination repair, impacts the response to chemotherapeutic agent olaparib, and exhibits aberrant patterns in subsets of human ovarian carcinomas.

Downregulation of BRCA1 protein in BCR-ABL1 leukemia cells depends on stress-triggered TIAR-mediated suppression of translation

BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3’UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins – HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3’UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 –positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in “BRCAness” CML and BCR-ABL1 –positive ALL cells.

5‐aminosalicylic acid interferes in the cell cycle of colorectal cancer cells and induces cell death modes

Introduction: Epidemiological data suggests that 5‐aminosalicylic acid (5‐ASA), a nonsteroidal antiinflammatory drug used in the treatment of inflammatory bowel diseases, prevents colorectal cancer development in these patients, although the mechanisms remain incompletely understood. Methods and Results: Here we report that 5‐ASA prevents growth of several colorectal cancer cell lines by interfering in the cell cycle, i.e., an S‐phase and G2/M phase arrest, dependent on 5‐ASA dosage and concentration, together with an increased mitotic index. In addition, prolonged cell cycle arrest by repeated 5‐ASA treatment induced apoptosis and caused abnormal spindle organization leading to mitotic catastrophe, another form of cell death. Conclusion: These observations illustrate that 5‐ASA has chemopreventive and chemotherapeutic properties. (Inflamm Bowel Dis 2010;)

Expression of Oncogenic Kinase Bcr-Abl Impairs Mitotic Checkpoint and Promotes Aberrant Divisions and Resistance to Microtubule-Targeting Agents

Recent findings showed that BRCA1, in addition to its role in DNA damage response, acts as an upstream regulator of genes involved in the mitotic checkpoint regulation, thus protecting against promotion of aberrant divisions and aneuploidy. Moreover, there is also an indication that the BRCA1 protein is downregulated in chronic myeloid leukemia (CML) patients. We have investigated a possible functional relationship between BRCA1 and mitotic checkpoint competence in cells with the same genetic background expressing different levels of Bcr-Abl, an oncogene responsible for CML. Herein, we show that Bcr-Abl strongly downregulates the BRCA1 protein level, which is partially reversed on treatment with imatinib, an inhibitor of Bcr-Abl tyrosine kinase. Bcr-Abl leads to decreased expression of genes involved in the mitotic checkpoint activation—Mad2, Bub1, Bub3, and BubR1, resulting in mitosis perturbances, weakened mitotic checkpoint function, and mitotic slippage after nocodazole treatment. Furthermore, high Bcr-Abl–expressing cells showed also postmitotic checkpoint dysfunctions and inability to effectively arrest in the 4NG1 phase of the cell cycle, which was associated with limited p21 induction. These observations had significant biological consequences, as we found a high level of improper divisions, chromosomal missegregation, and generation of polyploid cells on mitotic checkpoint prolonged activation. Additionally, Bcr-Abl–expressing cells showed resistance to death activated by spindle defects, reversed by imatinib. Our study presents new facts and supports the hypothesis concerning the mutator nature of Bcr-Abl itself. The functional interaction between Bcr-Abl and mitosis dysfunctions, due to compromised mitotic checkpoints, may have important implications for the generation of aneuploidy and CML progression. Mol Cancer Ther; 9(5); 1328–38. ©2010 AACR.

Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention.

BCR-ABL Hits at Mitosis; Implications for Chromosomal Instability, Aneuploidy and Therapeutic Strategy

1.1 Genomic and chromosomal instability in CML An unstable genome is a common hallmark of nearly all solid tumors and most of leukemias in contrast to normal, healthy cells which are able to maintein genome integrity (Negrini et al., 2010). Genomic instability could result from changes in chromosome structure and number as well as changes on the DNA level. Chromosomal instability (CIN) arises from unproper chromosome segregation as well as division defects and leads to aneuploidy (Foijer, 2010), whereas accumulation of mutations and DNA alterations usually is an effect of the defective repair systems and DNA damage response in cancer cells (Economopoulou et al., 2011). Chronic myeloid leukemia (CML) cells expressing the BCR-ABL tyrosine kinase have been found to accumulate mutations as well as chromosomal abnormalities. One of the first indications that CML correlates with additional chromosome changes has been presented in 1987 (Alimena et al., 1987). Moreover, authors showed that the rate of chromosomal anomalies increased during the blastic transformation. In the next years this has been also confirmed by other authors (Hagemeijer, 1987; Johansson et al., 2002; Su et al., 1999; Suzukawa et al., 1997). Later, random aneuploidy rate between chromosomes 9 and 18 has been reported in CML patients – both, untreated as well as upon imatinib therapy (Amiel et al., 2006). In broader analysis of CML patients it was found that chromosomal instability caused by centrosomal aberrations significantly correlated with the disease progression (Giehl et al., 2005). In the chronic phase only one sample out of 18 showed additional karyotypic alterations, in contrast to blast crisis where 73% patients (11/16) displayed additional karyotype alterations. The observation that CML patients have karyotype aberrations was confirmed in other studies where complex chromosomal rearrangements (CCR) were investigated (Babicka et al., 2006). By using cytogenetics, the FISH, and multicolor FISH (mFISH) methods, a very high level of the genomic instability at the chromosomal level, in cells obtained from chronic myeloid leukemia patients was observed. Altogether, it was shown that the aberrations associated with the progression of BCR-ABLpositive CML chronic phase to the aggressive blast crisis include additional chromosomes (Ph1, +8, +19), isochromosome 17q (associated with the loss of p53), reciprocal