Intisar Husain

A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer

The development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. Little is known about the mechanism(s) of acquired resistance to these agents. Here we establish a model of acquired resistance to N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib), an inhibitor of ErbB2 and ErbB1 tyrosine kinases by chronically exposing lapatinib-sensitive ErbB2-overexpressing breast cancer cells to lapatinib, simulating the clinic where lapatinib is administered on a daily chronic basis. Analysis of baseline gene expression in acquired lapatinib-resistant and parental cells indicates estrogen receptor (ER) signaling involvement in the development of resistance. Using gene interference, we confirm that acquired resistance to lapatinib is mediated by a switch in cell survival dependence and regulation of a key antiapoptotic mediator from ErbB2 alone to codependence upon ER and ErbB2 rather than loss of ErbB2 expression or insensitivity of ErbB2 signaling to lapatinib. Increased ER signaling in response to lapatinib is enhanced by the activation of factors facilitating the transcriptional activity of ER, notably FOXO3a and caveolin-1. Importantly, we confirm that lapatinib induces ER signaling in tumor biopsies from patients with ErbB2-overexpressing breast cancers receiving lapatinib therapy. These findings provided the rationale for preventing the development of acquired resistance by simultaneously inhibiting both ER and ErbB2 signaling pathways. Establishing clinically relevant models of acquired resistance to ErbB2 kinase inhibitors will enhance therapeutic strategies to improve clinical outcomes for patients with ErbB2-overexpressing breast cancers.

Plk3 Functionally Links DNA Damage to Cell Cycle Arrest and Apoptosis at Least in Part via the p53 Pathway

Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997)J. Biol. Chem. 272, 28646–28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029–6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylatedin vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3K52R) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

Lapatinib Antitumor Activity Is Not Dependent upon Phosphatase and Tensin Homologue Deleted on Chromosome 10 in ErbB2-Overexpressing Breast Cancers

Trastuzumab antitumor activity in ErbB2-overexpressing breast cancers seems to be dependent upon the presence of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphatase that dampens phosphatidylinositol 3-kinase-Akt signaling. Consequently, PTEN deficiency, which occurs in 50% of breast cancers, predicts for resistance to trastuzumab monotherapy. Here, we show that lapatinib, a small-molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, exerts its antitumor activity in a PTEN-independent manner. Steady-state phosphorylated ErbB2 (p-ErbB2) and p-Akt (S473) protein levels were inhibited within 30 min following lapatinib but not in response to trastuzumab in BT474 and Au565 cells (two ErbB2-overexpressing breast cancer cell lines that are sensitive to the proapoptotic effects of lapatinib). Whereas trastuzumab reportedly inhibits SRC phosphorylation (Y416), which in turn reduced SRC-ErbB2 protein interactions, lapatinib had no effect on either variable. To assess the potential functional role that PTEN might play in lapatinib antitumor activity, we selectively knocked down PTEN in BT474 and Au565 cells using small interfering RNA transfection. Loss of PTEN did not affect induction of tumor cell apoptosis by lapatinib in either cell line. In addition, lapatinib inhibited Akt phosphorylation in MDA-MB-468 cells, an ErbB1-expressing/ErbB2 non-overexpressing breast cancer line, despite their PTEN-null status. Moreover, patients with ErbB2-overexpressing inflammatory breast cancers responded to lapatinib monotherapy regardless of PTEN status. Thus, lapatinib seems to exert its antitumor activity in ErbB2-overexpressing breast cancers in a PTEN-independent manner. These data emphasize the importance of assessing PTEN status in tumors when selecting ErbB2-targeted therapies in patients with breast cancer.

Resistance to ErbB2 Tyrosine Kinase Inhibitors in Breast Cancer Is Mediated by Calcium-Dependent Activation of RelA

The widespread clinical use of therapies targeting the ErbB2 receptor tyrosine kinase oncogene represents a significant advance in breast cancer treatment. However, the development of therapeutic resistance represents a dilemma limiting their clinical efficacy, particularly small-molecule tyrosine kinase inhibitors that block ErbB2 autophosphorylation and activation. Here, we show that lapatinib (GW572016), a highly selective, small-molecule inhibitor of the ErbB2 and epidermal growth factor receptor tyrosine kinases, which was recently approved for the treatment of advanced-stage ErbB2+ breast cancer, unexpectedly triggered a cytoprotective stress response in ErbB2+ breast cancer cell lines, which was mediated by the calcium-dependent activation of RelA, the prosurvival subunit of NF-κB. Abrogation of lapatinib-induced RelA activation using either small interfering RNA constructs or an intracellular calcium chelator enhanced the apoptotic effects of lapatinib in parental ErbB2+ breast cancer cells and overcame therapeutic resistance to lapatinib in ErbB2+ breast cancer lines that had been rendered resistant to lapatinib through chronic exposure to the drug, mimicking the clinical setting. In addition, analysis of changes in phospho-RelA expression in sequential clinical biopsies from ErbB2+ breast cancers treated with lapatinib monotherapy revealed marginally statistically significant differences between responders and nonresponders, which was consistent with our preclinical findings. Elucidating the regulation of RelA by lapatinib in ErbB2+ breast cancers, and showing its role in the development of therapeutic resistance to lapatinib, identifies another therapeutic target to overcome or prevent the onset of resistance to lapatinib in some women with ErbB2+ breast cancers. Mol Cancer Ther; 9(2); 292–9

Photoreactivation of killing in E. coli K-12 phr− cells is not caused by pyrimidine dimer reversal

Abstract— Escherichia coliK–12 strains deleted in the phr gene which encodes the apoenzyme of photolyase show significant residual photoreactivation activity after exposure to high photoreactivating light doses. We show that this photoreactivation of killing is not associated with reversal of pyrimidine dimers and conclude that the effect is probably attributable to the direct photoconversion of the6–4 photoproducts to their Dewar isomers (Type III photoreactivation).

Cellular Functions of Mammalian DNA Ligases

DNA strand breaks, in particular double-strand breaks, are potentially cytotoxic lesions. These breaks may be introduced directly by a DNA-damaging agent, such as ionizing radiation, or as a consequence of DNA repair proteins recognizing and excising DNA damage. Thus, the majority of DNA repair mechanisms, including recombinational repair pathways, share a common essential step, phosphodiester bond formation, that restores the integrity of the DNA substrate molecule(s). In addition, DNA-joining events are required to link together the Okazaki fragments generated during lagging strand DNA synthesis.

In Vitro Repair of Cisplatin-Dna Adducts by a Defined Enzyme System

It is now well established that DNA is the target for the cytotoxic effects of platinum anticancer drugs (1). There are molecular mechanisms that remove the Pt adducts from DNA and thus reverse the biological effects of the Pt drugs. These DNA repair mechanisms have been characterized in E. coli. Of particular interest is the nucleotide excision because this pathway has been shown to be the major repair pathway responsible for cis-DDP resistance of this organism (2). Nucleotide excision repair in E. coli is mediated by ABC excision nuclease (excinuclease) which is composed of three subunits, the UvrA, UvrB and UvrC proteins. The enzyme incises on both sides of the modified nucleotide(s) hydrolyzing the 8th phosphodiester bond 5′ (7 bases 5′) and the 4th or 5th phosphodiester bond 3′ (3 or 4 bases 3′) to the adduct (3). Thus it removes a little over one turn of DNA from the damaged strand. The resulting gap is filled in by DNA polymerases and sealed by ligase.