Michael Clarke, MD

The Clarke laboratory reports the following updates on its work investigating the molecular pathways that regulate self-renewal and continues to work closely with other laboratories in identifying gene signatures of normal and cancer stem cells. These projects include ongoing and new research.

Breast Cancer

Identification of Cancer Stem Cells that Contain Low Levels of Reactive Oxygen Species and are Radioresistant
Investigators: Maximilian Diehn, Robert Cho, Neethan Lobo, Tomer Kalisky, Mary Jo Dorie, Angela Kulp, Dalong Qian, Jessica Lam, Laurie Ailles, Manzhi Wong, Benzion Joshua, Michael Kaplan, Irene Wapnir, Fred Dirbas, George Somlo, Carlos Garberoglio, Benjamin Paz, Jeannie Shen, Sean K. Lau, Stephen Quake, Martin Brown, Irving Weissman, and Michael Clarke

Metabolism of oxygen, while central to life, also produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease, and aging. It has recently been shown that central nervous system stem cells, hematopoietic stem cells, and early progenitors contain lower levels of ROS than their more mature progeny and that these differences appear to be critical for maintaining stem cell function. This team hypothesized that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. In this work, they showed that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Congruently, subsets of CSCs in some human and murine breast tumors contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing, radiation-induced cell killing, CSCs in these tumors develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacologic depletion of ROS scavengers in CSCs significantly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumors contain lower ROS levels and enhanced ROS defenses compared to their non-tumorigenic progeny, which may contribute to tumor radioresistance.

DLL4 Blockade Inhibits Tumor Growth through Multiple Mechanisms Including a Reduction of Cancer Stem Cell Frequency
Investigators: Timothy Hoey, Wan-Ching Yen, Fumiko Axelrod, Jesspreet Basi, Lucas Donigian, Scott Dylla, Maureen Fitch-Bruhns, Sasha Lazetic, In-Kyung Park, Aaron Sato, Sanjeev Satyal, Xinhao Wang, Michael Clarke, John Lewicki, and Austin Gurney

Previous studies have shown that blocking the signaling of the protein DLL4 reduced tumor growth by disrupting productive angiogenesis. These investigators developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. They found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes (for the Notch signaling pathway, a cell signaling system present in most multicellular organisms) and reduced proliferation of tumor cells. Furthermore, they found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells
Investigators: Yohei Shimono, Maider Zabala Ugalde, Robert Cho, Neethan Lobo, Piero Dalerba, Dalong Qian, Maximilian Diehn, Huiping Liu, Sarita Panula, Eric Chiao, George Somlo, Renee Reijo Pera, Kaiqin Lao, and Michael Clarke

The team has now published their finding that human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal adult and embryonic stem cells. Molecular regulators of self renewal and differentiation shared by normal and cancer stem cells have yet to be described.

  • These scientists found 37 miRNAs that were differentially expressed between BCSCs and their autologous non-tumorigenic cancer cells. Three clusters, miR-200c 141, miR-200b-200a-429, and miR-183-96-182 were down-regulated both in normal and breast cancer stem cells. MiR-200c and miR-183 inhibited the clonogenicity of breast cancer cells and suppressed the growth of embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. Most importantly, they showed that miR-200c expression strongly suppressed the outgrowth of normal mammary tissue and the tumor formation driven by human BCSCs in vivo. The down-regulation of these three miRNA clusters provides a molecular link that connects breast cancer stem cells to normal stem cells.

The Prognostic Role of a Gene Signature from Tumorigenic Breast Cancer Cells
Investigators: Rui Liu, Xinhao Wang, Grace Chen, Piero Dalerba, Austin Gurney, T. Hoey, Gavin Sherlock, J. Lewicki, K. Shedden, and Michael Clarke

This group discovered that breast cancers contain a minority population of cancer cells characterized by CD44 expression but low or undetectable levels of CD24 (CD44+CD24-/low) that have higher tumorigenic capacity than other subtypes of cancer cells. They have now published their findings on this subject.

  • These collaborators compared the gene-expression profile of CD44+CD24-/low tumorigenic breast-cancer cells with that of cells from normal breast epithelium. They used differentially expressed genes to generate a 186-gene invasiveness gene signature (IGS), which they evaluated for its association with overall survival and metastasis-free survival in patients with breast and other types of cancer. There was a significant association between the IGS and both overall and metastasis-free survival in patients with breast cancer, which was independent of established clinical and pathological variables. Combined with the prognostic criteria of the National Institutes of Health, they used the IGS to stratify patients with high-risk early breast cancer into prognostic categories (good or poor). Among patients with a good prognosis, the 10-year rate of metastasis-free survival was 81 percent; among those with a poor prognosis, it was 57 percent. The genetic signature of tumorigenic breast-cancer cells was even more strongly associated with clinical outcomes when combined with the wound-response (WR) signature in breast cancer.
  • The IGS was also associated with prognosis in medulloblastoma, lung cancer, and prostate cancer. As in breast cancer, the prognostic power of the IGS was increased when combined with the WR signature.

Blood Cancer

Dysregulated Gene Expression Networks in Human Acute Myelogenous Leukemia Stem Cells
Investigators: Ravindra Majeti, M. W. Becker, Q. Tian, M. Lee, Xinrui Yan, R. Liu, J. Chiang, Michael Clarke, L. Hood, and Irving Weissman

The investigators performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) with leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, they identified 3,005 differentially expressed genes. Using two independent analyses, they identified multiple pathways that are aberrantly regulated in leukemic stem cells in comparison to normal HSC. Several pathways, including Wnt signaling (in a network of proteins involved in embryogenesis and cancer), MAP kinase signaling (which regulates various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis), and adherens junction (for protein complexes at cell-cell junctions in epithelial tissues), are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations can yield an understanding of the critical pathways regulating cancer stem cells.