Date of Award

January 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Marc D. Basson

Abstract

Schlafens are a family of genes expressed in a wide range of mammals, including rodents and humans. Schlafens are grouped based on molecular structure and size into the short, intermediate, and long Schlafen families. Mice express all three families of Schlafens; in contrast, humans express only long and intermediate Schlafens. Schlafens have diverse functions, including cell proliferation, cell differentiation, immune cell growth and maturation, viral replication, cancer cell biology, and chemosensitivity. Our studies have focused on Schlafen 12 (SLFN12). SLFN12 is an intermediate Schlafen, it is expressed in humans but not in rodents, and is localized to the cytoplasm. SLFN12 lacks a c-terminal domain that contains the nuclear import signal that localizes the long Schlafens to the nucleus. SLFN12 induces differentiation in enterocytes, prostate cancer cells, sensitizes cancer cells to the apoptotic effect of phosphodiesterase inhibitors, estrogen, and nauclefine. The role of Schlafen 12 is poorly explored in cancer biology, in particular in lung and breast cancer. We used viral vectors and siRNA techniques to manipulate SLFN12 levels in cancer cells. To investigate the effect of manipulating SLFN12 levels in a series of lung and breast cancer cell lines, we utilized survival analysis assays, RT-qPCR, Western blot, flow cytometry, cell proliferation techniques, mammospheres formation assay, enzyme assay, and an in vivo xenograft mouse model.

In chapter one, we reviewed the Schlafen proteins in terms of structure, non-malignant functions, and the role of different Schlafens, including SLFN12 in different malignancies and chemosensitivity.

In chapter two, we demonstrated that SLFN12 correlates with survival in lung adenocarcinoma but not lung squamous cell carcinoma. Furthermore, SLFN12 reduced proliferation and induced apoptosis in lung adenocarcinoma cell lines but not lung squamous cell carcinoma cell lines. Mechanistically, we demonstrated that SLFN12 induces its antiproliferative effect by inhibiting c-myc translation in lung adenocarcinoma cells.

In chapter three, we demonstrated that SLFN12 correlates with survival of triple negative breast cancer patients. Furthermore, SLFN12 reduced the proliferation, invasion, cancer stem cells, mammospheres formation, and induced differentiation in triple negative breast cancer cells. We demonstrated that SLFN12 acts in part by translational inhibition of ZEB1 in triple negative breast cancer cells.

In chapter four, we demonstrated that SLFN12 delayed breast cancer tumorigenesis in vivo, increased tumor latency and slowed tumor growth. Moreover, SLFN12 prolonged the survival of animals injected with breast cancer cells overexpressing SLFN12. We identified that a L222A point mutation and a ∆150-333 deletion mutation abolished the antiproliferative effect and ZEB1 reducing effect of SLFN12 in triple negative breast cancer cells.

In chapter five, we identified Inosine-5′-monophosphate dehydrogenase-2 (IMPDH2) as a binding partner of SLFN12 and demonstrated that SLFN12 not only binds but also inhibits the activity of IMPDH2. Furthermore, we demonstrated that the inhibition of IMPDH2 activity by SLFN12 is another pathway through which SLFN12 induces its antiproliferative effect in triple negative breast cancer cells.

In conclusion, increased SLFN12 expression correlates with better survival in lung adenocarcinoma and triple negative breast cancer, and SLFN12 drives favorable effects by inducing the differentiation and reducing the aggressiveness of cancer cells through post-transcriptional regulation of proteins like c-myc and ZEB1 and the inhibition of IMPDH2 activity.

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