In the present study we observed that: (a) MLL2, primarily regarded as a nuclear protein with nuclear localization signals, displayed significant cytoplasmic localization in both normal and malignant cells, (b) mammary and colonic cell lines derived from highly invasive tumors exhibited altered sub-cellular distribution and proteolytic processing of MLL2 compared to non-tumor/less-invasive-tumor cell lines, and (c) MLL2 is overexpressed in breast and colon tumors tissues compared to the corresponding normal adjacent tissues.
MLL2 is primarily regarded as a nuclear protein. However, we observed both nuclear and cytoplasmic MLL2 in cell lines and tissue sections from breast and colon. In the breast epithelial cell lines, MLL2 specific bands representing the cleaved (75 kD) protein were observed in the cytoplasmic fractions of not only the tumor cell lines but also the non-tumor cell lines, 184A1 and MCF10a (Fig ); while bands representing the full-length, unprocessed MLL2 (290 kD) were present in the highly invasive tumor cell lines alone, and which may be related to oncogenic activity. The nuclear-cytoplasmic localization pattern of MLL2 was also evident in the benign sections of both breast and colonic tissues, but it was not possible to delineate the cleaved from the uncleaved MLL2 as the antibody used in this study cannot differentiate one from the other in situ
(details of antibody specificity are described in Methods section). Considering this cytoplasmic presence of MLL2 in the non-tumor cell lines and non-tumor tissue sections, it is possible that MLL2 may have a yet unidentified function in the cytoplasm, besides its role in epigenetic regulation in the nucleus [22
]. However, a notably increased cytoplasmic presence in the cancer cell lines and cancer tissues, together with the presence of MLL2FL
could be a tumor-related anomaly resulting from overexpression or increased protein/RNA stability.
Our study of the breast and colonic cell lines revealed two notable trends related to the proteolytic processing of MLL2. First, we observed a gradient in the intensities of the 75 kD signal in the nuclear fractions of both breast and colonic cell lines (Fig. &). In the breast epithelial cell lines, the intensities of the 75 kD nuclear MLL2, indicative of normal proteolytic processing, showed a decreasing trend with increasing malignancy from MCF10A to MDA-MB-231 except for MCF7. Of these cell lines, 184A1 and MCF10A are non-tumorigenic, MCF7 [16
] and T47D are tumorigenic [15
] but weakly invasive [24
], while MDA-MB-157 and MDA-MB-231 [16
] are highly invasive [15
]. The non-tumor cell line, 184A1, did not fit this trend and this discrepancy could be attributed to an overall lower level of MLL2 in 184A1, and/or the difference in proliferation rate--184A1 cells are reported to have a relatively lower proliferation rate compared to MCF10A [25
]. In the colonic epithelial cell lines a similar intensity gradient was noted for the 75 kD nuclear MLL2 fragment. Of these cell lines, HT29 [17
], DLD-1 [18
] and Ls174T [19
] are derived from well-differentiated tumors while, Lovo [18
], Colo205 [20
] and SW480 [21
] are derived from poorly differentiated tumors; and the 75 kD nuclear MLL2 was least in the Colo205 and SW480 cell lines.
The second notable trend was the presence of an additional band of 290 kD size (corresponding to the uncleaved precursor MLL2FL
) in the more invasive/poorly differentiated cell lines. More importantly, the MLL2FL
(290 kD) signal intensity increased in the nuclear and cytoplasmic fractions as the 75 kD nuclear signal decreased (Fig. &). This uncleaved MLL2FL
observed in the cytoplasmic and nuclear fractions of the advanced tumor cell lines could be a consequence of insufficient endogenous taspase 1 required for processing the excess MLL2. Since proteolytic cleavage could also be impaired as a consequence of non-cleavable mutations in MLL2FL
or the absence/decrease in the endogenous taspase 1 that cleaves MLL2 [2
], we looked for mutations in the MLL2
sequence coding for taspase 1 cleavage site, and also examined taspase 1 protein levels in the cell lines. Sequence analysis of the coding region for taspase 1 cleavage sites in cell lines carrying MLL2FL
did not reveal any alterations in the cleavage site encoding sequences. In addition, we found that the taspase 1 protein levels did not vary in parallel with the presence or absence of MLL2FL
. Taspase 1 protein levels were consistent across the six breast tissue cell lines, irrespective of the presence of MLL2FL
. Although we did not observe consistent levels of taspase 1 across the colonic cell lines, we did observe that the presence or absence of MLL2FL
and its levels, failed to correlate with taspase 1 level. That is, higher levels of taspase 1 did not correspondingly correlate with decreased levels of MLL2FL
or its total absence. These results suggest that the presence of MLL2FL
in the invasive cell lines is not a consequence of diminished levels of taspase 1 or a mutated cleavage site in the MLL2
. Further investigation is required, to determine the cause and consequences of MLL2FL
in the nucleus, which might be related to the shift in the nuclear-cytoplasmic localization of MLL2 in the invasive cell lines. Whatever may be the cause, our results suggest that the presence of precursor MLL2FL
is associated with a higher degree of malignancy.
According to an earlier report [2
], proteolytic processing of MLL2FL
is crucial to its stability, sub-nuclear localization, and methyltransferase activity. Impaired proteolytic maturation could result in significant changes in the normal epigenetic regulatory activities of MLL2. It has been shown in vitro
that MLL2 forms a multiprotein complex with Wdr5-Ash2L [26
] and associates with proteins like Pax7 and NF-E2 to direct histone lysine methylation at specific gene loci [3
]. MLL2 specific histone methylation complex is also known to associate with the tumor suppressor protein, menin, and mediate histone methylation at Hoxc8
]. Given the critical role for MLL2 in histone methylation activities, we believe that proteolytically immature and/or inappropriately expressed MLL2 may fail to effectively associate with the other members of the histone-methyltransferase complex, which in turn can adversely affect its role in epigenetic gene regulation.
In our analysis of the breast and colonic cell lines by reverse transcription (RT)-PCR, we also observed that MLL2 RNA levels were highest in the invasive tumor cell lines and least in the non-tumor/less-invasive-tumor cell lines (Fig. &). This trend in the MLL2 RNA levels was consistent with our observation of the overall increase in protein levels in the invasive tumor cell lines and in the tumor tissues. Since a real-time measurement was not performed on these cell lines it is not known if the increased levels of RNA resulted from an increased rate of transcription. The observed gradient in RNA levels in the breast and colon cell lines could also be due to differences in RNA stability. Though the RT-PCR results are not strictly quantitative, the results do indicate that MLL2 RNA levels are more abundant in the highly invasive/less differentiated cell lines.
Finally, our study on tissue sections from breast and colon cancer patients revealed that immunohistochemically detected MLL2 is significantly increased in tumor tissues. In breast tumors from patient samples, cytoplasmic MLL2 was significantly overexpressed as compared to normal adjacent tissues, and in colon tumors both cytoplasmic and nuclear MLL2 were significantly overexpressed when compared to adjacent benign mucosa. Since immunohistochemical signals can often arise from non-specific antibody reactions, we evaluated the specificity of antibody reactivity using a blocking peptide, a part of which represented the epitope recognized by anti-MLL2 antibody (detailed in Methods section). The peptide blocks the ability of the antibody to bind to its antigen. These experiments confirmed a high specificity of the antibody to the MLL2 antigen (Fig. ).
Analysis of MLL2 expression data with clinicopathological variables revealed a small correlation between MLL2 overexpression and early tumor stages (breast and colon) and absence of lymph node involvement (colon). However, the number of cases in each category was too small and the association too tentative to draw a substantive conclusion at this juncture. Despite a lack of correlation with established clinicopathological variables, the elevated levels of MLL2 protein in both breast and colon cancer was significant. These results are in line with our observation of increased MLL2 protein and RNA levels in the cell lines. However, due to the lack of an appropriate antibody to distinguish the cleaved MLL2 from uncleaved MLL2FL
by immunohistochemistry, we cannot comment on the composition of the overexpressed protein as to whether it constituted more of the 75 kD fragment or the 290 kD MLL2FL
. The elevated levels of MLL2 in the tumor tissues could be the result of overexpression, genomic amplification, increased RNA and/or protein stability, or, at least in part, due to alterations in protein processing. For example, genomic amplification of MLL2
(through complex chromosomal rearrangements or chromosomal duplications) resulting in four MLL2
copies has been previously reported in one of the breast cancer cell line, MDA-MB-157, used in this study [13
Whichever may be the cause, deregulated expression of MLL2 and/or defective proteolytic processing may adversely impact MLL2 mediated histone methylation activities, and in turn, disrupt downstream target genes potentially involved in cell cycle or cell proliferation activities. Consequently, aberrantly expressed MLL2 driven epigenetic regulation may contribute to tumor growth and/or progression. If such is the case, deregulation of MLL2 may be a more generic feature in tumorigenesis rather than an event specific to a particular tumor type, as is indicated by our findings in both breast and colon tumors.