Reactivation of the Inactive X Chromosome and Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Angela Andersen, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0075 Award: $48,563
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)

One of the two X chromosomes in females is transcriptionally silenced during embryogenesis in order to equalize X-linked gene dosage in females with that of XY males. The inactive X (Xi) is normally maintained throughout the female's lifetime; however, many human, female-specific cancers, including breast cancer, show loss of the Xi and a gain of active X (Xa) chromosomes, either through Xa duplication or Xi reactivation. There are over 70 cancer-related genes on the X, and the accumulation of multiple Xa chromosomes is functionally equivalent to the amplification of several genes shown to be involved in breast cancer initiation and metastasis. These observations suggest that the Xa may play a role in breast cancer.

We hypothesize that accumulation of multiple Xa chromosomes serves to expand a population of cancer stem/progenitor cells. Some mouse models of breast cancer, but not others, are associated with progenitor cell expansion. Thus, many questions remain. Which mouse models are associated with Xi reactivation? Is reactivation of the Xi tumorigenic in the mouse mammary gland? If Xi reactivation is not sufficient for tumorigenesis, can it cooperate with overexpression of the MYC oncogene to form mammary tumors? How does Xi reactivation alter proliferation and apoptosis of mammary epithelial cells?

Our aims in this project are as follows:

  1. Analyze different mouse models of breast cancer for reactivation of the Xi.
  2. Determine the effects of reactivation of the Xi in the mammary gland in whole animals.
  3. Investigate the mechanism by which Xi reactivation contributes to tumorigenesis.

For these studies we will use RNA fluorescent in situ hybridization (RNA FISH) to analyze the expression of X-linked genes in various mouse models of breast cancer that have been characterized as arising from progenitor cells (MMTV-Wnt-1) or from differentiated cells (MMTV-Neu, -PyMT). Next, we will isolate mouse mammary epithelial cells (MECs, including stem cells), genetically manipulate them to reactivate the Xi, transplant them into recipient mice, and analyze for early tumor-like outgrowths. If Xi reactivation is not sufficient for tumorigenesis, MECs with a reactivated Xi and excess amounts of MYC will be transplanted and analyzed. Finally, we will genetically and/or chemically reactivate the Xi in MECs and perform assays for cellular behaviors in cell culture conditions and study the genetic changes in these cells using microarray techniques.

Despite a correlation of Xi reactivation with breast cancers, more direct experimental studies have not been done. If Xi reactivation leads to breast cancer, understanding the molecular mechanism of reactivation and the pathways responsible for tumorigenesis is essential. In the future, therapies designed to re-inactivate additional Xa chromosomes may be helpful in devising new treatment strategies. In addition, new screening techniques for susceptibility and/or prognosis could be developed based on simple assays for the presence of multiple Xa chromosomes.




Final Report (2007)

Note: the PI resigned the grant early to take a position outside California.

In females, one of the two X chromosomes is inactivated early during embryogenesis to equalize X-linked gene dosage with XY males. Specific types of human breast cancer, including basal-like cancer (BLC), have acquired X chromosomal abnormalities such as the loss of the inactive X (Xi) and/or a gain of active X (Xa) chromosomes, either through Xa duplication or Xi reactivation. These abnormalities are associated with an increased expression of at least 30 X-linked genes, including some that have previously been shown to be involved in breast cancer. These observations suggest that deregulation of X inactivation may play a role in breast cancer.

Our initial aim was to analyze X inactivation in different mouse models of breast cancer. We performed RNA fluorescent in situ hybridization (RNA FISH) of X-linked genes on tumor cells derived from tumors that have been characterized as arising from an expansion of progenitor cells (MMTV-Wnt-1, -Myc) or of differentiated cells (MMTV-Neu, -PyMT). Xist is an X-linked gene expressed exclusively from the Xi; this non-coding RNA coats the Xi and plays a role in maintaining the silent state. Loss of Xist RNA enrichment from the Xi correlates with human BLC cancer. We found that most cells from each mouse model had Xist RNA coating a single Xi, and furthermore that genes normally subject to X inactivation were expressed exclusively from the single Xa. These data indicate that X inactivation is maintained in the different mouse models of breast cancer analyzed.

Although deregulation of X inactivation was not observed in the mouse models analyzed, it may still contribute to mammary tumorigenesis. To determine the consequence of de-repression of the Xi, we would need to isolate mouse mammary epithelial cells (MECs, including stem cells), genetically manipulate them to lose Xist expression and reactivate the Xi, and transplant them into recipient mice. Then, we would be a position to determine if Xi reactivation can cooperate with overexpression of the proto-oncogene c-Myc in tumorigenesis.