Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n ¼ 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3 0 untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention. Cancer Res; 73(15); 4872-84. Ó2013 AACR.

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<p>PDF - 492K, Supplementary Figure 1: Levels of the nine members of the let-7 family in pediatric malignant-GCTs. Supplementary Figure 2: PCR quantification of let-7e, LIN28 and LIN28B in malignant-GCTs.Supplementary Figure 3: Sylamer analysis of up-regulated genes in pediatric and adult malignant-GCTs. Supplementary Figure 4: Correlations between levels of let-7 and mRNA targets. Supplementary Figure 5: PCR quantification of HMGA2 in pediatric malignant-GCTs. Supplementary Figure 6: PCR validation of expression of selected let-7 mRNA targets in pediatric malignant-GCTs. Supplementary Figure 7: Effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 8: Independent confirmation of specific effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 9: Effects of let-7e mimic in malignant-GCT cells. Supplementary Figure 10: Relationships between MYCN and C-MYC versus LIN28 and LIN28B in malignant-GCTs. Supplementary Figure 11: MYCN depletion in malignant-GCTs (S11). Supplementary Figure 12: Schematic of the LIN28/let-7 axis in malignant-GCTs.</p>

<p>PDF - 62K, Supplementary Table 1: Clinico-pathological data for sample set-3. Supplementary Table 2: Primers used for mRNA qRT-PCR. Supplementary Table 3: Expression of let-7 family microRNAs in pediatric malignant-GCTs. Supplementary Table 4: Let-7 mRNA targets identified in combined microarray analysis of pediatric and adult malignant-GCTs. Supplementary Table 5: The six let-7 mRNA targets selected following microarray and qRT-PCR analysis.</p>

<p>PDF - 59K.</p>

<p>PDF - 62K, Supplementary Table 1: Clinico-pathological data for sample set-3. Supplementary Table 2: Primers used for mRNA qRT-PCR. Supplementary Table 3: Expression of let-7 family microRNAs in pediatric malignant-GCTs. Supplementary Table 4: Let-7 mRNA targets identified in combined microarray analysis of pediatric and adult malignant-GCTs. Supplementary Table 5: The six let-7 mRNA targets selected following microarray and qRT-PCR analysis.</p>

<p>PDF - 492K, Supplementary Figure 1: Levels of the nine members of the let-7 family in pediatric malignant-GCTs. Supplementary Figure 2: PCR quantification of let-7e, LIN28 and LIN28B in malignant-GCTs.Supplementary Figure 3: Sylamer analysis of up-regulated genes in pediatric and adult malignant-GCTs. Supplementary Figure 4: Correlations between levels of let-7 and mRNA targets. Supplementary Figure 5: PCR quantification of HMGA2 in pediatric malignant-GCTs. Supplementary Figure 6: PCR validation of expression of selected let-7 mRNA targets in pediatric malignant-GCTs. Supplementary Figure 7: Effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 8: Independent confirmation of specific effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 9: Effects of let-7e mimic in malignant-GCT cells. Supplementary Figure 10: Relationships between MYCN and C-MYC versus LIN28 and LIN28B in malignant-GCTs. Supplementary Figure 11: MYCN depletion in malignant-GCTs (S11). Supplementary Figure 12: Schematic of the LIN28/let-7 axis in malignant-GCTs.</p>