Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells

https://doi.org/10.1016/j.bmc.2019.115154Get rights and content

Abstract

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6β-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.

Introduction

Mitosis is a tightly regulated process of cell division that ensures the equal partition and transmission of genetic material to daughter cells. The characteristic dysregulation of cell division and proliferation in cancer cells have led to the use of mitotic inhibitors such as paclitaxel (taxol) and vinca alkaloids, which regulate microtubule dynamics, as anticancer agents.1 However, these drugs have many potential side effects due to the importance of microtubule formation in other processes, such as axonal transport in neuronal cells.2 Hence, the development of new anticancer agents that target mitosis specifically in cancer cells is highly desired.

Mitotic kinesins are a large family of proteins expressed strongly in cells during the M phase of cell cycle. These proteins regulate microtubule organization and dynamics and thus play a critical role in bipolar spindle formation. Previous research suggests that some mitotic kinesin inhibitors may be highly selective for cancer cells.3 For example, kinesin-5 Eg5, which exhibits plus-end directed motility, is the most well-evaluated potential drug target in this protein family. Research in this area revealed that the inhibition of Eg5 induces monopolar spindle formation, cell cycle arrest, and apoptosis in cancer cells. To date, several synthetic Eg5 inhibitors have been reported and assessed as monotherapies and/or combination therapies in clinical and non-clinical trials.4 However, these inhibitors also induce cytotoxicity in normal cells, which renders them less appealing as cancer therapeutic agents.

HSET/KIFC1 is a kinesin-14 family member with minus-end directionality.5 According to recent reports, HSET contributes to cancer cell survival via several mechanisms, particularly centrosome clustering. Many cancer cells, especially triple-negative breast cancers, contain supernumerary centrosomes due to mutations or deletions within oncogenes and/or tumor suppressor genes.6, 7 Although these cells would normally be directed to undergo cell death upon reaching the spindle assembly checkpoint, HSET enables the assembly of multiple centrosomes and the formation of pseudo-bipolar spindles. These features allow cancer cells to avoid apoptosis-induced cell death and thus exacerbate malignancy. In supernumerary centrosome-containing breast cancer cells, the knockdown of HSET prevents centrosome clustering and induces cell death via the formation of a multipolar spindle in anaphase. However, HSET knockdown is not lethal in normal cells that contain two centrosomes.8 Human breast adenocarcinoma cell line, MDA-MB-231, contained highly supernumerary centrosomes. An in vivo study reported that HSET knockdown inhibited xenograft tumor growth and suppressed centrosome clustering in mice.7, 8 HSET knockdown also inhibited the formation of gastric cancer cell spheroids and enhanced sensitivity to the clinical chemotherapeutic agents cisplatin and docetaxel.7, 9, 10 In contrast, HSET overexpression increased the steady-state levels of survivin, an apoptosis inhibitor protein, in cancer cells by decreasing poly-ubiquitination in a centrosome clustering-independent manner.11 These results highlight HSET as a promising drug for cancer therapy.

We previously demonstrated that in fission yeast cells, HSET overproduction induces the emergence of lethal monopolar spindles. Furthermore, our system of phenotypic screening for growth-restoring activity, which assesses the inhibition of HSET function using liquid medium or solid plate assays, appeared to be highly useful for the identification of HSET inhibitors.12 In this study, we used a simple plate assay to identify three natural products, solidagonic acid (SA) (1), kolavenic acid analog (KAA) (2) (a stereo isomer at C-9 and C-10 of 6β-tigloyloxykolavenic acid) and kolavenic acid (KA) (3) from Solidago altissima, which restored growth in HSET-overproducing yeast cells. All three compounds may inhibit HSET motor activity by promoting the conversion from abnormal monopolar to bipolar spindles. Furthermore, compound 2 inhibited centrosome clustering in human cancer cells containing high HSET levels and supernumerary centrosomes. These compounds appear to be the first natural products to restore the growth in a fission yeast screening system. Interestingly, the most potent compound, 2, which differs stereochemically from 1 and 3, may inhibit HSET in MDA-MB-231 cells. Therefore, compound 2 is expected to be a leading factor in the development of novel anticancer compounds and may be useful as a bioprobe for assessing modulation of the motor protein function.

Section snippets

Screening for compounds that could rescue growth in HSET-overproducing fission yeast cells

We previously demonstrated that HSET overproduction induced lethal growth defects in fission yeast cells, which could be rescued partially by treatment with AZ82, a synthetic HSET inhibitor.12, 13 Compounds found to restore the growth of these yeast cells may therefore possess HSET inhibitory activity and potentially could be used as an anti-tumor compound. Given this assumption, we screened SCADS Inhibitor Kits, and the inhibitors targeted tubulin or Eg5 using a simple plate assay [Fig. S1

Conclusion

HSET is a key factor in the survival of cancer cells containing supernumerary centrosomes. In this context, HSET contributes not only to centrosome clustering, but also to malignancy with centrosome amplification and therefore is a promising target for the selective inhibition of cancer cell proliferation. Here, we report three novel natural products isolated from Solidago altissima that were determined to restore the growth of HSET-overproducing fission yeast cells. All compounds induced the

Strains, cells, media, and genetic methods

The fission yeast strains used in this study were drug-sensitive strains (YA8; h+ bfr1::ura4+ pmd1::hisG leu1 ura4) containing the pREP41-GFP vector and pREP41-GFP-HSET plasmids (TY26 and TY27).27 The media, growth conditions, and manipulations were set as described previously.28, 29, 30 Rich YE5S liquid media and agar plates were used for most experiments. The YA8 strain was provided by Y. Yashiroda and M. Yoshida (Chemical Genetics Laboratory, RIKEN, Saitama, Japan). For overexpression

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgments

We are grateful to Ms. Shizuko Nakajo from the Center for Regional Collaboration in Research and Education of Iwate University for HRFABMS, to Dr. Yukio Kawamura for the introduction of a confocal laser microscope and to Dr. Yoko Yashiroda and Professor Minoru Yoshida of RIKEN for providing a fission yeast strain. We would like to thank Emeritus Professor Don R. Phillips of La Trobe University and Enago (www.enago.jp) for English language editing.

Funding sources

This work was partially supported by Iwate University, Japan (K.K.) and the Japan Society for the Promotion of Science, Japan, KAKENHI Scientific Research (A) under Grant [16H02503]; Challenging Exploratory Research under Grant [16K14672] (T.T.) and KAKENHI Scientific Research (C) under Grant [16K07694, 19K05813] (M.Y.).

Author contributions

K.K. designed the research and supervised the preparation of the manuscript. N.K., M.Y., H.K., and T.O. performed the experiments. T.T. read and corrected the draft. All authors reviewed the manuscript.

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