Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Synthesis and biological activities of topoisomerase I inhibitors, 6-arylmethylamino analogues of edotecarin.

The replacement of 1,3-dihydroxy-2-propylamino moiety at the N6-position of edotecarin (1) by arylmethylamino groups yielded a number of more potent topoisomerase I inhibitors with better cytotoxic (CTX) activities in vitro than edotecarin. Among them, the three most potent pyridylmethyl analogues, compounds 22g, 22m, and 23c, showed better antitumor activities against MKN-45 human stomach cancer or MX-1 human breast cancer xenografted mice than those of edotecarin. Furthermore, compounds 22m and 23c exhibited complete response against MX-1 cells implanted in mice.

ABCG2 confers resistance to indolocarbazole compounds by ATP-dependent transport.

The ABC half-transporter, ABCG2, is known to confer resistance to chemotherapeutic agents including indolocarbazole derivatives. MCF7 cells were introduced by either wild type ABCG2 (ABCG2-482R) or mutant ABCG2 (-482T), whose amino acid at position 482 is substituted to threonine from arginine, and their cross-resistance pattern was analyzed. Although this amino acid substitution seems to affect cross-resistance patterns, both 482T- and 482R-transfectants showed strong resistance to indolocarbazoles, confirming that ABCG2 confers resistance to them. For further characterization of ABCG2-mediated transport, we investigated indolocarbazole compound A (Fig. 1) excretion in cell-free system. Compound A was actively transported in membrane vesicles prepared from one of the 482T- transfectants and its uptake was supported by hydrolysis of various nucleoside triphosphates. This transport was inhibited completely by the other indolocarbazole compound, but not by mitoxantrone, implying that the binding site of mitoxantrone or the transport mechanisms for mitoxantrone is different from those of indolocarbazoles. These results showed that ABCG2 confers resistance to indolocarbazoles by transporting them in an energy-dependent manner.

Cystatin-like metastasis-associated protein mRNA expression in human colorectal cancer is associated with both liver metastasis and patient survival.

PURPOSE AND EXPERIMENTAL DESIGN: We previously reported that an increased expression of cystatin-like metastasis-associated protein (CMAP) mRNA is involved in liver-specific metastasis in a mouse model. We also identified its human homologue and showed that the expression of CMAP in various human cancer cell lines correlated with the description of malignancy in these cell lines. However, there is still no information available on the clinical significance of CMAP expression in human cancer specimens. Thus, we studied the CMAP expression levels using a real-time quantitative reverse transcription-PCR for 79 patients with colorectal cancer, including 17 cases with liver metastasis. RESULTS: The mean expression level of CMAP in tumor tissue specimens was significantly higher than in the corresponding normal tissue specimens (P < 0.05). A higher expression of CMAP was significantly correlated with liver metastasis (P < 0.01) as well as with a less differentiated histological type (P < 0.05) of colorectal cancer. An increased expression of CMAP was also identified as the strongest independent factor for liver metastasis based on a multivariate analysis (P < 0.001). Furthermore, the prognosis of the patients with a higher expression of CMAP was significantly worse than those with a lower expression (5-year survival rate; 49.7% and 75.0%, respectively, P = 0.038). CONCLUSIONS: These findings imply that the expression level of CMAP in human cancer may be a new biomarker for both liver metastasis and the patient's outcome.