siRNA Targeting of Thymidylate Synthase, Thymidine Kinase 1 and Thymidine Kinase 2 as an Anticancer Therapy: A Combinatorial RNAi Approach

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS siRNA to sensitize tumour cells to TS-targeting drugs. Downregulation of TKs with siRNA enhanced the capacity of TS siRNA to sensitize tumour cells to traditional TS protein-targeting drugs (5FUdR and pemetrexed). Combined downregulation of these enzymes is an attractive strategy to enhance TS-targeted anticancer therapy. TK2 can phosphorylate both thymidine and deoxycytidine to generate dTMP and dCMP, precursors for dTTP and dCTP, respectively. dCTP negatively regulates deoxycytidine kinase (dCK), another enzyme that phosphorylates deoxycytidine as well as the anticancer drug gemcitabine. Antisense knockdown of TK2 could reduce TK2-produced dCMP, thus decreasing dCTP levels and inhibition of dCK, and lead to increased dCK activity, gemcitabine activation, and anticancer effectiveness. Given the substrate promiscuity of TK2, we hypothesized that: (1) TK2 can mediate human tumour cell resistance to gemcitabine, (2) antisense downregulation of TK2 can overcome that resistance, and (3) TK2 siRNA-induced drug sensitization results in mitochondrial damage. siRNA downregulation of TK2 expression sensitized MCF7 and HeLa cells to gemcitabine, but did not sensitize A549 cells (low TK2 expresser). Treatment with TK2 siRNA and gemcitabine: 1) decreased mitochondrial redox status, 2) decreased mitochondrial DNA (mtDNA:nDNA ratio), and 3) decreased mitochondrial activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 from other dTMP-producing enzymes

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency invivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent

IDO Downregulation Induces Sensitivity to Pemetrexed, Gemcitabine, FK866, and Methoxyamine in Human Cancer Cells.

Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy

A549 clone sensitivity to methoxyamine (3 mM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</b>) IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Cultured medium was then replaced with fresh growth medium containing Methoxyamine (MX) (3 mM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 3 for determination of each value) ± SD. Results are normalized to control cells not treated with methoxyamine, without (panel A) or with (panel B) IFNγ treatment. <b>Panel C</b>: Induction of IDO in A549 clonal cell populations induces resistance to MX (3 mM). Results were obtained from 3 or 2 independent clonal cell populations with scrambled, non-targeting control shRNA or anti-IDO shRNA, respectively. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: Relationship between IDO protein level (relative to actin) and resistance to methoxyamine (MX)(proliferation relative to untreated control cells). The R² value of 0.83 represents a moderate positive relationship.</p

TS siRNA downregulation in A549 clonal populations.

<p>A549 clonal cell populations (NC-3, NC-10, and NC-30, each with stably-incorporated control, non-targeting scrambled shRNA; and 2–6 and 2–18, each with stably-incorporated anti-IDO shRNA) were seeded and grown overnight. Thymidylate synthase (TS) siRNA numbers 3 or 4, or scrambled, non-targeting control siRNA, were transiently transfected into each clonal population. Cells were lysed and protein was harvested 96 h later. TS and actin protein levels were determined by immunoblot. Results were quantified for each clone separately. <b>A</b>: TS protein quantification for each clonal population. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SEM. *Significant difference from the same cells transfected with scrambled control, non-targeting siRNA, Student's <i>t</i>-test, <i>p</i><0.05. <b>B</b>: Representative immunoblots of TS and actin protein in clone NC-3 after transfection of TS siRNA (siRNA numbers 3 and 4) or scrambled, non-targeting control siRNA (C2). <b>C</b>: Representative immunoblots of TS and actin protein in clone 2–18 after transfection of TS siRNA.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to pemetrexed more than IDO knockdown alone.

<p>A549 cells were transfected with scrambled, non-targeting control siRNA or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, pemetrexed (30 nM) for 72, and then enumerated. Bars indicate mean relative cell numbers (n = 3 ± SD). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with pemetrexed, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing scrambled, non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with scrambled, non-targeting control siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with pemetrexed. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were each considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with pemetrexed, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

A549 clonal cell population sensitivity to FK866 (5 nM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</b>) IDO induction with IFNγ. A549 clonal cell populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Medium was then replaced with fresh growth medium containing FK866 (5 nM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Results are normalized to control cells not treated with FK866, without (panel A) or with (panel B) IFNγ treatment. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SD, (*, p≤0.05). <b>Panel C</b>: A549 clonal resistance to FK866 before and after IFNγ-mediated IDO induction. Pooled results were obtained from 3 or 2 independent A549 clonal populations stably transfected with scrambled shRNA (white bars) or 2 anti-IDO shRNA (black bars) respectively, before and after induction of IDO. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: Correlation analysis of the relationship between IDO protein content (relative to actin) and clonal population resistance to FK866 (proliferation relative to untreated control cells).</p

A549 clone sensitivity to pemetrexed (200 nM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before <b>(Panel A)</b> and after <b>(Panel B)</b> IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h, then with pemetrexed (200 nM), and enumerated 72 h later. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SD. Results are normalized to control cells not treated with pemetrexed, without (panel A) or with (panel B) IFNγ treatment. <b>Panel C</b>: Induction of IDO in A549 clonal cell induces resistance to pemetrexed (200 nM). Results were obtained from 3 or 2 independent clonal cell populations with scrambled, non-targeting control shRNA or anti-IDO shRNA, respectively. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to 5FUdR more than TS knockdown alone.

<p>A549 cells were transfected with control or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, with 5FUdR (40 nM) for 72 h, and then enumerated. Bars indicate mean proliferation relative to appropriate controls ± SD (n = 3). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with 5FUdR, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with control non-targeting siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with 5FUdR. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with 5FUdR, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

A549 clone sensitivity to Gemcitabine and 5FUdR before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before <b>(Panel A and C)</b> and after <b>(Panel B and C)</b> IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h, treated with gemcitabine (10 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels A and B and SEM for panel C, (*<i>p</i><0.05). Results are normalized to control cells not treated with Gemcitabine, without (panel A) or with (panel B) IFNγ treatment. <b>Panels D-F</b>: Proliferation of each of 5 individual A549 cell clonal populations before and after IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h and, 5FUdR (200 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels D and E and SEM for panel F ± SD. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Results are normalized to control cells not treated with 5FUdR, without (panel A) or with (panel B) IFNγ treatment.</p