Xuefeng Liu - US grants

DESCRIPTION (provided by applicant): Cell immortalization is a critical event in the development of cancer and the oncogenic human papillomaviruses encode an E6 oncogene that is primarily responsible for inducing telomerase activity and consequent immortalization. Recent studies indicate that the HPV E6 protein mediates the increase in telomerase activity via its ability to engage and activate the hTERT promoter and interact directly with at least two promoter-bound proteins, Myc and NFX-1. E6 also has a post-translational mechanism to increase telomerase activity by binding directly to hTERT proteins. In the current grant, we show that hTERT immortalization of primary keratinocytes is independent of its telomerase activity or ability to elongate telomeres. In agreement with recent work with stem cells, our new preliminary findings demonstrate that hTERT can alter the expression of keratinocyte genes as well as increase the expression of HPV genes. We hypothesize that the transcription activation function of hTERT or its potential role in altering apoptotic responses may play an essential role in bypassing the M1/M2 restriction points in cell proliferation and thereby facilitate cell immortalization. In this application, we will evaluate these possibilities using a library of mutan hTERT expression vectors. We will correlate the ability of these mutant proteins to efficiently bypass cellular senescence with their ability to induce reverse transcriptase activity, hTR binding, telomere binding, telomere elongation, promoter transactivation, and modulation of apoptosis. These studies will provide new insights into a basic event in the etiology of cancer.

Prostate cancer (PCa) is the most frequently diagnosed cancer (180,890 new cases in 2016) in men in the USA. Androgen deprivation therapy (ADT) is an effective first line therapy for locally advanced or metastatic disease. Unfortunately, once PCa recurs, the eventual development of castration-resistant prostate cancer (CRPC) remains an incurable disease and more effective therapies are needed. Currently, a limited number of cancer cell lines (LnCAP, PC3, DU-145, etc.) are available for research and many genetic mutations present in prostate cancer (e.g., SPOP mutation, FOXA1 mutation, TMPRSS2-ERG fusion, CHD1 loss) are not represented in such cells. New patient-derived cancer models are needed. However, patient-derived xenograft (PDX) models are successful at less than 2-5% efficiency with aggressive, high-grade metastatic tumors and organoid cultures only have an efficiency of 20%. However, our preliminary data demonstrate that conditional reprogramming (CR) has nearly a 100% success rate for establishing long-term cultures from either surgical prostate specimens or CT-guided biopsies. In this application, we propose the following specific aims to validate the potential of CR for translational use in human CRPC. We will first establish CR cultures from biopsies of 30 patients with CRPC and will characterize these culture genetically and phenotypically. Second, we compare the patients' drug response to those of corresponding tumor CR cells and their derivative CR- derived xenografts (CDXs). Lastly, we will use CR cultures in an unbiased high-throughput screen to identify new potential therapies for CRPC in collaboration with Dr. Craig Thomas at National Center for Advanced Translational Sciences (NCATS). New ?hits? from the screen will be validated by both in vitro cell assays and xenograft models.

PROJECT SUMMARY Bladder cancer (BC) is the costliest cancer (per case) among all cancer types and yet among the top 10 cancers it is the most underfunded cancer by NCI. There has been no significant improvement in overall survival and prognosis over the last thirty years except for the recent development of immunotherapy. At initial diagnosis, approximately 75% of cases are diagnosed at the non-muscle-invasive stages, and are usually treated with transurethral resection (TUR) followed by intravesical instillation of therapeutic agents into the bladder cavity in high risk patients. This approach is associated with cancer recurrence of over 60% at two years and progression into advanced stages in up to 25% of patients. Therefore, almost all patients will need long-term expensive cystoscopy. If a sensitive but less expensive method to detect cancer were available, it would improve the treatment outcomes and decrease the cost. For locally advanced BC, neoadjuvant chemotherapy is associated with a complete response of less than 40%. For metastatic BC, the response rate for the first-line platinum-based chemotherapy is approximately 50%. It is less than 20% for second-line chemotherapy. Currently no method is available to predict which patients will respond to therapy. Liquid biopsies are non-invasive methods that may be applicable for cancer precision medicine. Circulating factors, including circulating tumor cells (CTCs), cfDNAs, RNAs (miRNAs, long non-coding RNAs [lncRNAs], mRNAs), cell-free proteins, peptides, or exosomes et al., are derived from cells in human body. However, it is still unclear where and at what tumor stage these circulating molecules are coming from. There are still too many technical issues that limit the study of CTC biology and their applications. Our latest data show that we are able to generate conditionally reprogrammed cell cultures (CRC) from urine samples of 60 BC patients. Therefore, we will focus on CR technology for generating urine-derived cultures to predict efficacy of treatment and recurrence of BC. We will also determine if urine-derived cell cultures are a simpler and potentially more sensitive technique to monitor bladder cancer recurrence. Urine samples from 70-90 BC cases (prior to initial transurethral resection and chemotherapy) will be used to generate bladder cancer cultures. Following therapy, the patients will be monitored every 3 months (for a total of 2 years) by cystoscopy, and urine cell culture. By comparing the detection of recurrence by each of these techniques, we can determine whether cell culture and/or mutant hTERT detection can provide sufficient sensitivity and specificity to replace the expensive and invasive cystoscopy procedure. We will also use cell cultures derived from pre-surgery patients to evaluate for sensitivity to standard of care drugs and compared with the clinical response of the patient to these same drugs. Last, we will use CR cultures from non-responding patients or recurrent patients in unbiased high-throughput screening to identify and validate new potential therapies for BC.