We describe a number of observations that suggest that cancer cells may differ from normal cells in their requirements for Thoc1. Thoc1
expression is higher in oncogene transformed human and mouse fibroblasts compared to normal fibroblasts. This mimics the increased Thoc1
expression observed in primary human breast cancer compared to normal mammary epithelium(4
), and the abundant levels of pThoc1 typically observed in human cancer cell lines that have been tested(5
). However, higher pThoc1 levels are not merely a reflection of the faster proliferation rate typical of cancer cells. Loss of pThoc1 in cancer cell lines and oncogene transformed human or mouse fibroblasts inhibit cell accumulation through induction of apoptosis and subsequent loss in cell viability. In contrast, loss of pThoc1 in normal human or mouse fibroblasts has no detectable effect on viability. Thus the requirements for Thoc1
to support viability are different in the tested normal and cancer cells. We have been unable to recover viable, E1A/Ha-ras
transformed or spontaneously immortalized MEFs that lack pThoc1. Thus cells undergoing neoplastic transformation or immortalization are unable to adapt to the absence of Thoc1
, suggesting Thoc1
may be required.
There are potential caveats in interpreting these experiments. Since we cannot recover viable E1A/Ha-ras expressing cells that lack pThoc1, we cannot exclude the possibility that pThoc1 loss inhibits neoplastic transformation by compromising expression of E1A/Ha-ras or the drug resistance genes. However, experiments have been performed in the absence of drug selection with similar results. E1A/Ha-ras transduction of Thoc1F/− MEFs, but not Cre pre-treated Thoc1F/− MEFs lacking pThoc1, generate cultures of immortal, transformed cells without drug selection (Y. Li and A. Lin, unpublished observation). Thus the inability to recover transformed cells in the absence of pThoc1 is independent of drug selection. In addition, MEFs lacking pThoc1 are viable, yet we do not recover viable cells upon transduction of these cells with E1A/Ha-ras. This suggests that E1A/Ha-ras must be expressed sufficiently in the absence of pThoc1 to trigger a biological response. We note that pThoc1 loss also inhibits spontaneous immortalization of MEFs and the viability of many different human cancer cell lines. The effects of Thoc1, therefore, are not specific to E1A/Ha-ras transduction and likely reflect a general requirement for Thoc1 in cancer cells.
We have previously observed that Thoc1
is required for the viability of blastocyst stage mouse embryos(23
). Blastocyst stage embryos are comprised largely of stem cells that, like cancer cells, have an extended potential for replication and self-renewal. Thus, Thoc1
may be required more generally for the maintenance of extended replicative potential. Consistent with this hypothesis, we have observed that primary MEFs genetically ablated for Thoc1
, while viable and able to proliferate in culture, undergo premature conversion to a senescence-like state (Y. Li and D.W. Goodrich, unpublished observation). Similarly, loss of the yeast orthologue HPR1
is not lethal, but compromises lifespan(29
). It is conceivable that lack of sufficient pThoc1 limits replicative potential in normal cells by induction of a cellular senescence program, thereby inhibiting immortalization and neoplastic transformation. Upregulation of Thoc1
may be required, therefore, to facilitate immortalization and neoplastic transformation. Since cancer cells are unable to sustain cell viability upon acute loss of pThoc1 activity, the effects of pThoc1 loss must be dominant to the effects of deregulated E1A/Ha-ras
expression studied here, as well as to other alterations that facilitate neoplastic transformation in a variety of different human cancer cell lines tested here.
loss triggers apoptosis in cancer cells is unknown. Since all detectable pThoc1 is apparently within the TREX complex(6
), the simplest explanation is that loss of pThoc1 compromises TREX function. Loss of TREX function could have several conceivable effects on cells. Loss of TREX activity may adversely affect the generation of translatable mRNA from a subset of genes required to maintain viability. Loss of TREX activity may also compromise normal telomere maintenance. In yeast, loss of the Thoc1
is associated with defects in telomere maintenance(30
). Defects in telomere maintenance would be expected to influence replicative potential and viability. Alternatively, deficient mRNP biogenesis in the absence of Thoc1
may trigger R loop formation and DNA strand breaks(31
). Such DNA lesions could trigger apoptotic cell death in cancer cells if they are unable to efficiently repair them. The accumulation of phosphorylated histone H2AX upon pThoc1 depletion observed here is consistent with this possible mechanism, but additional studies will be required to identify and verify the mechanism underlying loss of cancer cell viability.
Irrespective of the precise mechanism, mutational inactivation of Thoc1
is synthetic lethal with the genetic and epigenetic alterations associated with a number of cancer cell lines of different type and origin. Thus Thoc1
may represent a novel molecular target for cancer therapy. Therapy that blocks pThoc1 activity is expected to preferentially compromise the viability of cancer cells, potentially yielding superior therapeutic index. Since the mechanism of pThoc1 action is novel, utilization of pThoc1 as a therapeutic target may yield unique clinical responses and opportunities for novel combination therapy. For example, yeast deficient in the Thoc1
are synthetic lethal with topoisomerase mutations(32
) and are more sensitive to DNA damage(33
). Depletion of pThoc1 in human cancer cell lines renders them more sensitive to camptothecin and cisplatin(5
). These observations suggest that therapeutic inhibition of pThoc1 in human cancer will increase sensitivity to topoisomerase poisons and possibly other forms of genotoxic therapy.
protein functions in the newly discovered TREX complex, a representative of a class of complexes that regulate gene expression subsequent to transcriptional initiation. This class of protein complexes may specify post-transcriptional “operons” that facilitate protein expression from coordinately regulated genes of diverse size and structure(34
). Although there is increasing appreciation for the importance of such complexes, they are understudied relative to the transcription factors that govern the initiation of transcription. As such, their relevance to carcinogenesis is largely undocumented. However, the von Hippel-Lindau tumor suppressor protein is a known inhibitor of the elongin transcription elongation factor(35
). The interaction of the retinoblastoma tumor suppressor protein and pThoc1 may reflect an analogous interaction between a tumor suppressor gene product and a transcription elongation/RNA processing factor. Such interactions suggest that complexes that regulate gene expression at the level of mRNP formation and RNA processing may provide a largely untapped source of novel molecular targets for cancer therapy.