Targeting the Cell Cycle, RRM2 and NF-κB for the Treatment of Breast Cancers.

A hallmark of cancer is the dysregulation of the cell cycle. The CDK4/6 inhibitor palbociclib is approved for treating advanced estrogen-receptor-positive breast cancer, but its success is limited by the development of acquired resistance owing to long-term therapy despite promising clinical outcomes. This situation necessitates the development of potential combination strategies. Here, we report that didox, an inhibitor of ribonucleotide reductase in combination with palbociclib, can overcome palbociclib resistance in ER-positive and ER-negative breast cancers. This study shows didox downregulates an element of the cell cycle checkpoint, cyclin D1, accompanied by a reduction in NF-κB activity in vitro and tumor growth inhibition of palbociclib-resistant ER positive breast cancer tumor growth in vivo. Furthermore, didox induces cell cycle arrest at G1 as well as reduces ROS generated by on-target effects of palbociclib on the cell cycle. Our current study also reports that the CCND1 and RRM2 upregulation associated with palbociclib-resistant breast cancers decreases upon ribonucleotide reductase inhibition. Our data present a novel and promising biomarker-driven combination therapeutic approach for the treatment of ER-positive and ER-negative breast cancers that involves the inhibition of the CDK4/6-cyclinD1/pRb cell cycle axis that merits further clinical investigation in human models.

Molecular Targeting of RRM2, NF-κB, and Mutant TP53 for the Treatment of Triple-Negative Breast Cancer.

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 heart failure when used at cumulative doses exceeding 400 mg/m2 Previously, we have observed that the ribonucleotide reductase subunit M2 (RRM2) is significantly overexpressed 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, a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NF-κB activity in vitro When didox was used in combination with doxorubicin, we observed significant downregulation of NF-κB proteins accompanied by reduced TNBC cell proliferation. 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 synergistic 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 levels. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans.

Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents.

The hydrophobicity of many chemotherapeutic agents usually results in their nonselective passive distribution into healthy cells and organs causing collateral toxicity. Ligand-targeted drugs (LTDs) are a promising class of targeted anticancer agents. The hydrophilicity of the targeting ligands in LTDs limits its nonselective passive tissue distribution and toxicity to healthy cells. In addition, the small size of LTDs allows for better tumor penetration, especially in the case of solid tumors. However, the short circulation half-life of LTDs, due to their hydrophilicity and small size, remains a significant challenge for achieving their full therapeutic potential. Therefore, extending the circulation half-life of targeted chemotherapeutic agents while maintaining their hydrophilicity and small size will represent a significant advance toward effective and safe cancer treatment. Here, we present a new approach for enhancing the safety and efficacy of targeted chemotherapeutic agents. By endowing hydrophobic chemotherapeutic agents with a targeting moiety and a hydrophilic small molecule that binds reversibly to the serum protein transthyretin, we generated small hydrophilic drug conjugates that displayed enhanced circulation half-life in rodents and selectivity to cancer cells. To the best of our knowledge, this is the first demonstration of a successful approach that maintains the small size and hydrophilicity of targeted anticancer agents containing hydrophobic payloads while at the same time extending their circulation half-life. This was demonstrated by the superior in vivo efficacy and lower toxicity of our conjugates in xenograft mouse models of metastatic prostate cancer.

Pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolines: Novel compounds that reverse ABCG2-mediated resistance in cancer cells.

Overexpression of ATP-binding cassette transporter (ABC) subfamily G2 in cancer cells is known to elicit a MDR phenotype, ultimately resulting in cancer chemotherapy failure. Here, we report, for the first time, the effect of eight novel pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline (IND) derivatives that inhibit ABCG2 transporter restoring cancer cell chemosensitivity. IND -4, -5, -6, -7, and -8, at 10 µM, and nilotinib at 5 µM, significantly potentiated (8-10 fold) the cytotoxicity of the ABCG2 substrates mitoxantrone (MX) and doxorubicin in HEK293 cells overexpressing ABCG2 transporter, MX (~14 fold) in MX-resistant NCI-H460/MX-20 small cell lung cancer, and of topotecan (~7 fold) in S1-M1-80 colon cancer cells which all stably expressing ABCG2. In contrast, cytotoxicity of cisplatin, which is not an ABCG2 substrate, was not altered. IND-5,-6,-7, and -8 significantly increased the accumulation of rhodamine-123 in multidrug resistant NCI-H460/MX-20 cells overexpressing ABCG2. Both IND-7 and -8, the most potent ABCG2 inhibitors, had the highest affinities for the binding sites of ABCG2 in modeling studies. In conclusion, the beneficial actions of new class of agents warrant further development as potential MDR reversal agents for clinical anticancer agents that suffer from ABCG2-mediated MDR insensitivity.

Targeting Ribonucleotide Reductase M2 and NF-κB Activation with Didox to Circumvent Tamoxifen Resistance in Breast Cancer.

Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.

Genetic ablation of PRAS40 improves glucose homeostasis via linking the AKT and mTOR pathways.

Alterations in PI3K-AKT-mTOR signaling have been implicated in diabetes. This study assessed whether disruption of PRAS40, a substrate of AKT and component of mTORC1, would alter glucose homeostasis and prevent hyperglycemia in the streptozotocin (STZ)-induced diabetes mouse model. PRAS40 ablation resulted in a mild lowering of blood glucose levels and glycated hemoglobin (HbA1C), a lowered insulin requirement, and improved glucose tolerance in untreated PRAS40 gene knockout (PRAS40(-/-)) as compared to wild-type (PRAS40(+/+)) mice. Diabetes was then induced in these mice using STZ at 50mg/kg/day over five days. Following STZ-treatment, PRAS40(-/-) mice exhibited significantly lower blood glucose and HbA1C levels than PRAS40(+/+) mice. Liver tissue of PRAS40(-/-) mice and shPRAS40 Hep3B cells showed increased activation of AKT (p-AKT T308) and mTORC1 (p-p70S6K) signaling as well as decreased p-AKT (S473) and increased p-IRS1 (S612) protein levels. Altered tissue gene expression of several glucose transporters (GLUT) and increased hepatic GLUT4 protein levels were observed in PRAS40(-/-) as compared to PRAS40(+/+) mice. In summary, PRAS40 deletion significantly attenuates hyperglycemia in STZ-induced PRAS40(-/-) mice through increased hepatic AKT and mTORC1 signaling, a lowered serum insulin requirement, and altered hepatic GLUT4 levels.

Proline-rich AKT substrate of 40-kDa (PRAS40) in the pathophysiology of cancer.

Dysregulation of PI3K-AKT-mTOR pathway has been reported in various pathologies, such as cancer and insulin resistance. The proline-rich AKT substrate of 40-kDa (PRAS40), also known as AKT substrate 1 (AKT1S1), lies at the crossroads of these cascades and inhibits the activity of the mTOR complex 1 (mTORC1) kinase. This review discusses the role of PRAS40 and possible feedback mechanisms, and alterations in AKT/PRAS40/mTOR signaling that have been implicated in the pathogenesis of tumor progression. Additionally, we probed new datasets extracted from Oncomine, a cancer microarray database containing datasets derived from patient samples, to further understand the role of PRAS40 (AKT1S1). These data strongly supports the hypothesis that PRAS40 may serve as a potential therapeutic target for various cancers.

AKT-induced tamoxifen resistance is overturned by RRM2 inhibition.

UNLABELLED: Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)-mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis. IMPLICATIONS: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies.

Estrogen, tamoxifen, and Akt modulate expression of putative housekeeping genes in breast cancer cells.

Clinically, Akt overexpression has been associated with tamoxifen resistance, and multiple in vitro breast cancer models of tamoxifen resistance have been developed. In order to study the mechanism of this tamoxifen resistance, differential gene expression studies have been performed utilizing quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Since accurate data normalization requires the use of a stable reference gene, the goal of this study was to identify the most stable reference gene for RT-qPCR (from a panel of putative housekeeping genes) that remains unaltered despite estrogen or tamoxifen treatment or stable overexpression of active Akt. Gene expression of nine candidate genes was determined in parental and Akt overexpressing MCF-7 breast cancer cells treated with estrogen, tamoxifen, or vehicle, and gene stability was analyzed using two different statistical models. Based on our results, we suggest RPL13A as suitable internal reference gene that is both stable and remains unaltered in MCF-7 cells regardless of estrogen or tamoxifen treatment or Akt overexpression. We also validated that expression levels for RPL13A, as well as RPLP0 (another member of the RPL protein family), remain unaltered after estrogen and tamoxifen treatment in the ER positive ZR-75-1 cell line and ER negative MDA-MB-468 breast cancer cell line. Both RPL13A and RPLP0 levels were also stable in normal and tumor mammary tissue from Her2 overexpressing mice. In addition, our work emphasizes the importance of a preliminary study to validate each reference gene that will be used for RT-qPCR.

Anti-tumor pyrimidinylpiperazines bind to the prosurvival Bcl-2 protein family member Bcl-XL.

Overexpression of prosurvival or underexpression of pro-death Bcl-2 family proteins can lead to cancer cell resistance to chemotherapy and radiation treatment. Inhibition of the prosurvival Bcl-2 family proteins has become a strategy for cancer therapy and inhibitors are currently being evaluated in the clinic both as single agents and in combination with established drugs. Here we describe the design, synthesis, and evaluation of pyrimidylpiperazines that were discovered to be inhibitors of the prosurvival Bcl-2 protein family member Bcl-XL. This study identified compound 21 which demonstrated a GI(50) value of 8.4 µM against A549 lung adenocarcinoma cells and a binding affinity K(i) value for Bcl-XL of 127 nM.

Investigation on the Interactions of NiCR and NiCR-2H with DNA.

We report here a biophysical and biochemical approach to determine the differences in interactions of NiCR and NiCR-2H with DNA. Our goal is to determine whether such interactions are responsible for the recently observed differences in their cytotoxicity toward MCF-7 cancer cells. Viscosity measurement and fluorescence displacement titration indicated that both NiCR and NiCR-2H bind weakly to duplex DNA in the grooves. The coordination of NiCR-2H with the N-7 of 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) is stronger than that of NiCR as determined by (1)H NMR. NiCR-2H, like NiCR, can selectively oxidize guanines present in distinctive DNA structures (e.g., bulges), and notably, NiCR-2H oxidizes guanines more efficiently than NiCR. In addition, UV and (1)H NMR studies revealed that NiCR is oxidized into NiCR-2H in the presence of KHSO(5) at low molar ratios with respect to NiCR (

A truncated human Ah receptor suppresses growth of human cervical tumor xenografts by interfering with hypoxia signaling.

We used a xenograft model to investigate whether the aryl hydrocarbon receptor deletion construct CDelta553 suppresses tumor growth. HeLa cells that were infected with CDelta553 expressing adenovirus (Ad553) formed very small tumors whereas the control adenovirus-infected cells formed large tumors at day 15. CDelta553 inhibited the formation of the HIF-1 DNA complex and suppressed the induction of the HIF-1alpha target proteins CAIX and GLUT1. The Ad553 tumors had less HIF-1 function since they showed reduced microvessel formation and lesser amounts of HIF-1alpha, Arnt, phospho-Akt, CAIX, and GLUT1. Proteasome-mediated Arnt degradation was enhanced in Ad553-infected HeLa cells and tumors.

Modulation of cathepsin D routing by IGF-II involves IGF-II binding to IGF-II/M6P receptor in MCF-7 breast cancer cells.

The IGF-II/M6P receptor targets cathepsin D to the lysosomes and it also binds IGF-II. Although the binding sites for IGF-II and cathepsin D are distinct, reciprocal interactions between the ligands have been observed. We have demonstrated that proIGF-II expression modulates routing of cathepsin D. To test the hypothesis that IGF-II modulation of cathepsin D routing in MCF-7 cells involves IGF-II binding to the IGF-II/M6P receptor, we expressed a mutant form of IGF-II (Arg54 Arg55) that does not bind the IGF-II/M6P receptor and evaluated its effects on cathepsin D secretion. Northern blotting, Western and radioimmunoassay analyses confirmed that these cells express high levels of (Arg54 Arg55) IGF-II mRNA and secretes high levels of IGF-II without modulating the secretion of cathepsin D. These data provide direct evidence that the IGF-II modulation of cathepsin D routing is IGF-II/M6P receptor mediated.