TGFß-blockade uncovers stromal plasticity in tumors by revealing the existence of a subset of interferon-licensed fibroblasts.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFß in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFß and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor.

The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR's ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.

Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies.

A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

Contribution of polycomb homologues Bmi-1 and Mel-18 to medulloblastoma pathogenesis.

Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.

Insulin-like growth factor-binding protein 3 expression increases during immortalization of cervical keratinocytes by human papillomavirus type 16 E6 and E7 proteins.

Human papillomaviruses (HPVs) infect cervical epithelial cells and induce both benign and precancerous lesions. High-risk HPVs promote the development of cervical cancer in vivo and can immortalize cervical epithelial cells in vitro, whereas low-risk HPVs cannot. We used cDNA microarrays and quantitative polymerase chain reaction to compare cellular gene expression in primary cervical epithelial cells during a time course after retroviral transduction with either low-risk or high-risk E6/E7 genes. At early passages, cervical cells transduced with high-risk E6/E7 genes demonstrated increased expression of the cell cycle-regulated genes CDC2 and ubiquitin carrier E2-C. At later passages, these same cells exhibited dramatic increases in insulin-like growth factor-binding protein-3 (IGFBP-3) mRNA and both secreted an intracellular protein, with mRNA levels increasing approximately 85-fold. Corroborating these in vitro studies, in situ hybridization of cervical biopsies with an IGFBP-3 riboprobe revealed high levels of expression in high-grade squamous intraepithelial neoplasia but not in normal cervical epithelium. Our in vitro results indicate that overexpression of IGFBP-3 is a late event after E6/E7 expression, and analysis of cervical lesions indicates that overexpression of this gene is also seen in vivo.

Measurements of human papillomavirus transcripts by real time quantitative reverse transcription-polymerase chain reaction in samples collected for cervical cancer screening.

Specific assays capable of distinguishing normal and atypical cervical changes from pre-cancerous lesions are direly needed to improve screening for cervical cancer. Specific genes transcripts that are up-regulated in dysplastic and cancer cells can be exploited as new markers for cervical cancer screening provided that they can be detected in heterogeneous populations such as those collected for Papanicolaou tests. We hypothesized that expression of the HPV early region gene E7 might distinguish between normal samples (absent expression) and high-grade lesions (detectable E7 expression). Our goal was to detect and measure gene expression in cells scraped from the cervix using real time quantitative reverse transcription-polymerase chain reaction (TaqMan). We have optimized collection and extraction procedures to provide suitable RNA for TaqMan analysis in clinical samples collected for cervical cancer screening and have demonstrated efficient measurements of housekeeping genes in these samples. HPV 16 or 18 early gene E7 transcripts were detected in 47% of samples with a clinical diagnosis of high-grade SIL and in 0% of cytologically normal samples (P = 0.006). Our study demonstrates that the TaqMan assay can be reliably applied to samples collected for cervical cancer screening, and that presence of detectable HPV E7 transcripts can distinguish between normal and abnormal samples.