Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Mayo Clinic

Effectiveness of DNA cross-linking drugs in the treatment of bladder cancer suggests that bladder cancer cells may have harbored an insufficient cellular response to DNA cross-link damage, which will sensitize cells to DNA cross-linking agents. Cell sensitivity benefits from deficient DNA damage responses, which, on the other hand, can cause cancer. Many changed cellular signaling pathways are known to be involved in bladder tumorigenesis; however, DNA cross-link damage response pathway [Fanconi anemia (FA) pathway], whose alterations appear to be a plausible cause of the development of bladder cancer, remains an under-investigated area in bladder cancer research. In this study, we found FAVL (variant of FA protein L--FANCL) was elevated substantially in bladder cancer tissues examined. Ectopic expression of FAVL in bladder cancer cells as well as normal human cells confer an impaired FA pathway and hypersensitivity to Mitomycin C, similar to those found in FA cells, indicating that FAVL elevation may possess the same tumor promotion potential as an impaired FA pathway harbored in FA cells. Indeed, a higher level of FAVL expression can promote the growth of bladder cancer cells in vitro and in vivo, which, at least partly, results from FAVL perturbation of FANCL expression, an essential factor for the activation of the FA pathway. Moreover, a higher level of FAVL expression was found to be associated with chromosomal instability and the invasiveness of bladder cancer cells. Collectively, FAVL elevation can increase the tumorigenic potential of bladder cancer cells, including the invasive potential that confers the development of advanced bladder cancer. These results enhance our understanding the pathogenesis of human bladder cancer, holding a promise to develop additional effective tools to fight human bladder cancer.

FAVL impairment of the Fanconi anemia pathway promotes the development of human bladder cancer.

Fanconi Anemia (FA) complementation group D2 protein (FANCD2) is the center of the FA tumor suppressor pathway, which has become an important field of investigation in human aging and cancer. Here we report an overlooked central player in the FA pathway, FANCD2 variant 2 (FANCD2-V2), which appears to perform more potent tumor suppressor-function compared to the known variant of FANCD2, namely, FANCD2-V1. Detailed analysis of the FANCD2 gene structure indicated a proximal and distal polyadenylation site (PAS), associated with V2 and V1 transcripts accordingly. RNA polymerase II Chromatin immunoprecipitation (ChIP) targeting the two PAS-regions determined lesser binding of RNA pol II to DNA fragments in the distal PAS region in non-malignant cells compared to malignant cells. Conversely, the opposite occurred in the proximal PAS region. Moreover, RNA immunoprecipitation (RIP) identified that U2 snRNP, a major component of RNA splicing complex that interacts with the 3'end of an intron, showed greater binding to the last intron of the FANCD2-V1 transcript in malignant cells compared to the non-malignant cells. Importantly, our data showed that in human tissue samples, the ratio of V2 /V1 expression in lung, bladder, or ovarian cancer correlates inversely with the tumor stages/grades. Therefore, these findings provide a previously unrecognized central player FANCD2-V2 and thus novel insights into human tumorigenesis, and indicate that V2/V1 can act as an effective biomarker in assisting the recognition of tumor malignance.

Overlooked FANCD2 variant encodes a promising, portent tumor suppressor, and alternative polyadenylation contributes to its expression.

The mechanism of prostate cancer (PCa) progression towards the hormone refractory state remains poorly understood. Treatment options for such patients are limited and present a major clinical challenge. Previously, δ-catenin was reported to promote PCa cell growth  and its increased level is associated with PCa progression  In this study we show that re-arrangements at Catenin Delta 2 (CTNND2) locus, including gene duplications, are very common in clinically significant PCa and may underlie δ-catenin overexpression. We find that δ-catenin in PCa cells exists in a complex with E-cadherin, p120, and α- and β-catenin. Increased expression of δ-catenin leads to its further stabilization as well as upregulation and stabilization of its binding partners. Resistant to degradation and overexpressed δ-catenin isoform activates Wnt signaling pathway by increasing the level of nuclear β-catenin and subsequent stimulation of Tcf/Lef transcription targets. Evaluation of responses to treatments, with androgen receptor (AR) antagonist and β-catenin inhibitors revealed that cells with high levels of δ-catenin are more resistant to killing with single agent treatment than matched control cells. We show that combination treatment targeting both AR and β-catenin networks is more effective in suppressing tumor growth than targeting a single network. In conclusion, targeting clinically significant PCa with high levels of δ-catenin with anti-androgen and anti β-catenin combination therapy may prevent progression of the disease to a castration-resistant state and, thus, represents a promising therapeutic strategy.

Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists.

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using a whole-genome mate-pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS-OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti-HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2-targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo-resistant lines. When chemotherapy and HER2-targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post-treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS-OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2-targeted therapy.

Piyan Zhang

Center for Individualized Medicine

Mayo Clinic

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

Piyan Zhang

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Mayo Clinic

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

Center for Individualized Medicine