Use of constitutive and inducible oncogene-containing iPSCs as surrogates for transgenic mice to study breast oncogenesis.

BACKGROUND: Powerful constitutive and inducible transgenic / bitransgenic / tritransgenic murine models of breast cancer have been used over the past two decades to shed light on the molecular mechanisms by which the given transgenic oncogenes have interacted with other cellular genes and set in motion breast cancer initiation and progression. However, these transgenic models, as in vivo models only, are expensive and restrictive in the opportunities they provide to manipulate the experimental variables that would enable a better understanding of the molecular events related to initial transformation and the target cell being transformed. METHODS: To overcome some of these limitations, we derived oncogene-containing induced pluripotent stem cell (iPSC) clones from tail vein fibroblasts of these transgenic mice and manipulated them both in vitro and in vivo in non-transgenic background mice. We created the iPSC clones with a relatively low M.O.I, producing retroviral integrations which averaged only 1 to 2 sites per retroviral plasmid construct used. RESULTS: Most iPSC clones derived from each group displayed an essentially normal murine karyotype, strong expression of the exogenous reprogrammable genes and significant expression of characteristic endogenous murine surface stem cell markers including SSEA-1 (CD15), PECAM-1 (CD31), Ep-Cam (CD326), and Nectin (CD112), but no expression of their transgene. A majority (75%) of iPSC clones displayed a normal murine karyotype but 25% exhibited a genomically unstable karyotype. However, even these later clones exhibited stable and characteristic iPSC properties. When injected orthotopically, select iPSC clones, either constitutive or inducible, no longer expressed their exogenous pluripotency reprogramming factors but expressed their oncogenic transgene (PyVT or ErbB2) and participated in both breast ontogenesis and subsequent oncogenesis. When injected non-orthotopically or when differentiated in vitro along several different non-mammary lineages of differentiation, the iPSC clones failed to do so. Although many clones developed anticipated teratomas, select iPSC clones under the appropriate constitutive or inducible conditions exhibited both breast ontogenesis and oncogenesis through the same stages as exhibited by their transgenic murine parents and, as such, represent transgenic surrogates. CONCLUSIONS: The iPSC clones offer a number of advantages over transgenic mice including cost, the ability to manipulate and tag in vitro, and create an in vitro model of breast ontogeny and oncogenesis that can be used to gain additional insights into the differentiated status of the target cell which is susceptible to transformation. In addition, the use of these oncogene-containing iPSC clones can be used in chemopreventive studies of breast cancer.

Why is cancer so common a disease in people yet so rare at a cellular level?

Cancers are common diseases in people and yet, on a cellular level, are quite rare. The vast majority of both sporadic, spontaneous cancers and inherited germline cancers arise in single foci from singly transformed cells despite the fact that, in the former, carcinogenic factors bathe fields of millions of potential target cells and, in the latter, the predisposing germline mutations are present in every cell of a given organ and, in fact, every cell of the body. Although the multi-hit theory of carcinogenesis has been invoked to explain such things as cancer latency, which is the period between cancer initiation and emergence and the cancer-aging relationship where an accumulation of "hits" over a period of time are necessary for cancer emergence, the multi-hit theory falls short in explaining the rareness of transformation at a cellular level. This is so because many cancers are not due to multiple hits, and even for those that are, it would be expected that many cells would be exposed to those factors inducing the hits. Although the tumor stem/progenitor cell compartmental theory of tumorigenesis characterizes a tumor compartment that is capable of self-renewal and multipotency, accounting for cancer relapses and recurrences, this compartmental theory alone cannot account for the rareness of initial transformation at a cellular level as the cancer stem/progenitor cell compartment is already transformed and considerable in size. This study advances a different and novel hypothesis that oncogenesis is regulated and ultimately determined by a cell of origin's critical state of differentiation. Before and after this critical state of differentiation has been reached, target cells cannot transform and give rise to cancer even when they receive the necessary carcinogenic insults or have the requisite transforming tumor suppressor genes or oncogenes. As support for this hypothesis, the study cites preliminary evidence using oncogene-containing transgenic mice that develop mammary carcinomas, to derive tail vein fibroblasts converted to iPSCs which, when left undifferentiated, and injected into the cleared fat pads of non-transgenic background mice give rise to mammary gland ontogeny and mammary gland carcinogenesis. However, when first differentiated in vitro into multiply different non-mammary lineages prior to injection, they fail to do so. The hypothesis has widespread implications for chemopreventive strategies.

Regulation of breast cancer oncogenesis by the cell of origin's differentiation state.

Human breast cancer which affects 1/8 women is rare at a cellular level. Even in the setting of germline BRCA1/BRCA2, which is present in all breast cells, solitary cancers or cancers arising at only several foci occur. The overwhelming majority of breast cells (109-1012 cells) resist transformation. Our hypothesis to explain this rareness of transformation is that mammary oncogenesis is regulated by the cell of origin's critical window of differentiation so that target cells outside of this window cannot transform. Our novel hypothesis differs from both the multi-hit theory of carcinogenesis and the stem/progenitor cell compartmental theory of tumorigenesis and utilizes two well established murine transgenic models of breast oncogenesis, the FVB/N-Tg (MMTV-PyVT)634Mul/J and the FVB-Tg (MMTV-ErbB2) NK1Mul/J. Tail vein fibroblasts from each of these transgenics were used to generate iPSCs. When select clones were injected into cleared mammary fat pads, but not into non-orthotopic sites of background mice, they exhibited mammary ontogenesis and oncogenesis with the expression of their respective transgenes. iPSC clones, when differentiated along different non-mammary lineages in vitro, were also not able to exhibit either mammary ontogenesis or oncogenesis in vivo. Therefore, in vitro and in vivo regulation of differentiation is an important determinant of breast cancer oncogenesis.