Targeting ribonucleotide reductase for breast cancer treatment

Author

Nahid Sultana, University of the PacificFollow

Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Jesika S. Faridi

First Committee Member

Mamoun Alhamadsheh

Second Committee Member

Roshanak Rahimian

Third Committee Member

Atefeh Rabiee

Fourth Committee Member

Khyati N. Shah

Abstract

Breast cancer is the second most common type of cancer in the world. Hormone receptor (HR) positive breast cancer (BC) is a prevalent disease accounting for approximately 2 million new cases globally. Almost 70-80% of breast cancer patients are women with a positive score for the estrogen receptor (ER). Triple-negative breast cancer (TNBC) which have a negative score for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) is considered an aggressive histological breast cancer subtype with limited treatment options.Frequently, doxorubicin (DOXO)-based chemotherapy is utilized in this patient population due to the lack of available molecular targets. While DOXO is an effective chemotherapeutic agent, its efficacy is limited due to acquired drug resistance and cardiotoxicity. Therefore, the identification of other treatment options for TNBC is needed. TNBC is a heterogeneous malignancy, with 70% of cases classified as a basal subtype as they look similar to the epithelial cells of the outermost basal layer of the breast’s milk ducts. This further complicates the search for an effective molecular target. Doxorubicin and other anthracycline derivatives are frequently used as part of the adjuvant chemotherapy regimen for triple- negative breast cancer (TNBC). Although effective, doxorubicin is known for its off-target and toxic side effect profile, particularly with respect to the myocardium, often resulting in left ventricular (LV) dysfunction and congestive 2 heart failure when used at cumulative doses exceeding 400 mg/m2.

Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Both RRM1 and RRM2 are encoded by different genes in their chromosomes. Their mRNAs are also differentially expressed throughout the cell cycle. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. We have observed that the ribonucleotide reductase subunit 2 (RRM2) is significantly over-expressed in estrogen receptor (ER)–negative cells as compared with ER-positive breast cancer cells. Here, we inhibited RRM2 in ER-negative breast cancer cells as a target for therapy in this difficult-to-treat population. We observed that through the use of didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NFkB activity in vitro. When the ribonucleotide reductase inhibitor didox was used in combination with the chemotherapeutic drug doxorubicin, we observed significant downregulation of NFkB proteins in TNBC. As well, we observed that protein levels of mutant p53 were significantly reduced by didox or combination therapy in vitro. Xenograft studies showed that combination therapy was found to be effective in vivo, resulting in a significantly reduced tumor volume as compared with doxorubicin monotherapy. In addition, the use of didox was also found to ameliorate the toxic myocardial effects of doxorubicin in vivo as measured by heart mass, LV diameter, and serum troponin T protein levels which are released by heart during muscle damage. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans. Hormone receptor positive breast cancers of all stages are selectively treated with endocrine therapy targeting estrogen receptor (ER) activity. But success is limited by the development of acquired resistance owing to long-term therapy. The cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led to the development of the first CDK4/6 inhibitor Palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Intrinsic and acquired drug resistance development, have impacted the therapeutic success rate despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. The combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple-negative breast cancer. In our recent study, we confirmed that didox in combination with palbociclib significantly lowers the growth of ER positive and ER negative breast cancer cells along with their palbociclib resistant counterparts compared to no treatment or palbociclib treatment alone. We confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells exhibit lower IC50 values of palbociclib drug as compared to their palbociclib resistant counterparts. Here, we are reporting that didox alone or in combination with palbociclib decreases cell cycle proteins in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment alters cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer cells along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data present a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2, NFkB, and the CDK4/6-cyclin D1/pRb axis that merits further clinical investigation in human models.

Pages

97

Recommended Citation

Sultana, Nahid. (2023). Targeting ribonucleotide reductase for breast cancer treatment. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/4174