To express a gene to make a protein, the gene's DNA must first be transcribed to produce molecules of messenger RNA. The start of the transcription process features two milestones. First, an enzyme called RNA Polymerase II starts the process. Shortly afterwards, however, the process pauses and only starts again when other proteins are recruited. This second step, called transcriptional elongation, is essential for gene expression in cells that are growing and specializing into specific cell types. However, it is unclear how important this second step is for the progression of human cancers, such as breast cancer.

In humans, two proteins join together to form a complex called ‘positive transcription elongation factor b’ (or P-TEFb for short). This elongation factor encourages the transcriptional elongation step by adding phosphate groups onto RNA Polymerase II and by outcompeting other proteins that act to stop the process. However, some of the P-TEFb proteins in the cell's nucleus are unable to do this because they are held within a complex, which also contains an RNA molecule and some other proteins including one called LARP7. This protein–RNA complex is thought to help to prevent a number of cancers, for example breast cancer or stomach cancer; however the effect of P-TEFb proteins on cancers in humans is not known.

Less LARP7 protein is made in breast cancer cells compared to healthy cells. And when Ji et al. reduced the levels of the LARP7 protein (or the RNA molecule involved in the complex), the P-TEFb proteins were released from the complex and were free to encourage transcriptional elongation. This led to the increased expression of other proteins that switch other genes on or off, including genes that allow breast cancer cells to spread around the body. On the other hand, Ji et al. revealed that freeing the P-TEFb proteins from the complex in the nucleus did not appear to cause new tumors to develop or existing tumors to grow.

Ji et al. suggest that the LARP7 protein normally helps to prevent the spread of breast cancers by keeping the P-TEFb proteins inactive as a part of the protein–RNA complex. One of the next challenges will be to see if drugs that can inhibit the P-TEFb proteins might be useful as new treatments for late stage breast cancer.

Cancer cells have fundamentally altered gene expression profiles that drive their pathogenic features. In eukaryotes, gene transcription is mainly performed by RNA polymerase (Pol) II and can be controlled at multiple stages including pre-initiation, initiation, elongation, and termination (Shilatifard et al., 2003; Sims et al., 2004). Transcription elongation has recently been demonstrated to play a critical role in regulating cell growth and differentiation. In Drosophila and human embryonic stem cells, a large number of genes involved in cell growth, renewal, and differentiation are found to be controlled at the elongation stage (Guenther et al., 2007; Muse et al., 2007; Zeitlinger et al., 2007). Moreover, in mammalian cells, the transcription elongation machinery has also been implicated in the regulation of cell proliferation and differentiation (Zhou and Yik, 2006; Romano and Giordano, 2008).

Shortly after initiation of transcription, Pol II pauses near the transcription start site largely due to the actions of two negative transcription elongation factors NELF and DSIF (Peterlin and Price, 2006). The human positive transcription elongation factor b (P-TEFb) reverses this block and stimulates transcriptional elongation by phosphorylating the two negative elongation factors as well as the C-terminal domain (CTD) of the largest subunit of Pol II. These modification events antagonize the actions of the negative elongation factors and also promote co-transcriptional mRNA processing (Zhou et al., 2012). P-TEFb is a heterodimer composed of cyclin-dependent kinase 9 (CDK9) and its regulatory partner Cyclin T1 or T2 (CycT1 or T2). The activity of P-TEFb is stringently maintained in a functional equilibrium in cells to accommodate transcriptional demands for different biological activities (Zhou and Yik, 2006). Three P-TEFb-containing complexes have been identified, including the inhibitory 7SK snRNP complex and two active transcription complexes: the Brd4-P-TEFb complex and the super elongation complex (SEC) (Zhou et al., 2012; Lu et al., 2013b).

Under normal growth conditions, more than half of nuclear P-TEFb is sequestered in the catalytically inactive 7SK snRNP, which also contains the 7SK snRNA, HEXIM1 (or the homologous HEXIM2), MePCE, and LARP7. This complex represents the major cellular reservoir of inactive P-TEFb (Zhou and Yik, 2006; Zhou et al., 2012). Within 7SK snRNP, the 7SK snRNA serves as a central scaffold that coordinates key protein–protein interactions and allows HEXIM1/2 to inhibit CDK9 (Yik et al., 2003). The La-related protein LARP7 binds to nearly all the nuclear 7SK snRNA via the 3′-UUU-OH sequence and protects it against exonuclease cleavage (He et al., 2008; Krueger et al., 2008). Stable RNAi-mediated knockdown of LARP7 causes an almost complete depletion of 7SK snRNA, leading to disruption of 7SK snRNP and a shift of P-TEFb toward the active state (He et al., 2008). Considering that the 7SK snRNP is the primary source of suppressed P-TEFb, the level of LARP7 directly affects the amount of active P-TEFb, thereby playing a key role in controlling P-TEFb activity.

In response to a number of conditions/agents that globally impact growth and differentiation, P-TEFb is released from the 7SK snRNP and recruited to chromatin templates by the bromodomain protein Brd4, which binds to acetylated histones and the Mediator complex to promote transcription and cell cycle progression (Yang et al., 2005; Mochizuki et al., 2008; Yang et al., 2008). In addition to Brd4, P-TEFb released from 7SK snRNP can also be recruited by a number of gene-specific transcription factors such as the HIV-1 Tat protein and the mixed lineage leukemia (MLL) fusion proteins to form the multi-subunit SEC complex to specifically and efficiently activate their target genes (He et al., 2010; Sobhian et al., 2010; Lu et al., 2013a).

Accumulating data have implicated the involvement of various components of P-TEFb-containing complexes in human cancer. For example, several components of the P-TEFb-containing SEC, such as AFF1, AFF4, ELL1, AF9, and ENL, are all translocation partners of MLL and are important for MLL-based leukemogenesis (Lin et al., 2010; Yokoyama et al., 2010; Smith et al., 2011). In addition, Brd4 has also been considered a promising therapeutic target in acute myeloid leukemia (AML) because of its ability to sustain P-TEFb-dependent c-Myc expression (Blobel et al., 2011; Zuber et al., 2011). Finally, several lines of evidence have implicated the control of P-TEFb by the 7SK snRNP in human breast cancer. First of all, HEXIM1 has been proposed as an inhibitor of breast cell growth since its expression is downregulated by estrogens in breast tumors (Wittmann et al., 2003). Moreover, microsatellite instability (MSI)-induced frameshift mutations in the LARP7 gene have been detected in a significant population of gastric cancer samples, implicating a potential tumor suppressor role of LARP7 in cancers (Mori et al., 2002). Consistent with this result, we have previously shown that LARP7 knockdown in the mammary epithelial cell line MCF10A disrupts cell polarity and blocks morphological differentiation when cultured in the three-dimensional laminin-rich extracellular matrix (3D IrECM) (He et al., 2008).

Despite these observations, virtually nothing is known about whether P-TEFb and its associated factors may play a key role during human cancer progression. In this study, we investigated the function of the P-TEFb functional equilibrium in controlling the epithelial–mesenchymal transition (EMT), invasion, and metastasis of human breast cancer. By knocking down LARP7, we released P-TEFb from the 7SK snRNP and stimulated the P-TEFb-dependent transcription of EMT-related genes, resulting in breast cancer EMT and enhanced invasion and metastasis. Our analyses have revealed a strong causative relationship between the invasive phenotypes of human breast cancer and P-TEFb activation by disrupting the 7SK snRNP. Our study has thus provided the first demonstration that the transcription elongation machinery and the P-TEFb network play critical roles in regulating tumor progression, EMT, and metastasis by directly controlling the expression of EMT/metastasis-related genes.

In human cells, the 7SK snRNP represents the principle cellular reservoir of uncommitted P-TEFb, and its integrity is maintained through LARP7's direct interaction with and stabilization of the 7SK snRNA (He et al., 2008). Here, we show that in noninvasive human breast cancer cells, disruption of this complex by knocking down LARP7 releases P-TEFb, redistributing it to the transcriptionally active SEC complex. This activation of P-TEFb promotes breast cancer EMT, invasion, cancer stem cell expansion, and metastasis by directly activating the expression of genes involved in EMT and metastasis. Thus, the sequestration of P-TEFb by 7SK snRNP is an effective anti-cancer mechanism, and the key 7SK snRNP component LARP7 is a potential tumor suppressor that specifically blocks breast cancer progression and metastasis. Our data highlight the importance of the transcription elongation machinery in regulating breast cancer EMT and metastasis and suggest a new therapeutic option to combat metastatic breast cancer through blocking P-TEFb activation. Currently, the CDK9 inhibitor flavopiridol is being evaluated in several phase I and II clinical trials for its anti-cancer effects either as a single agent or in combination with other drugs in treatment of esophageal cancer, B-cell chronic lymphocytic leukemia, endometrial carcinoma, recurrent/metastatic squamous cell carcinoma and most relevantly, previously treated locally advanced or metastatic breast cancer (http://www.cancernetwork.com/review-article/current-clinical-trials-flavopiridol/page/0/2). Thus, our studies are particular relevant and provide the mechanistic basis for targeting P-TEFb in patients with metastatic breast cancer.

LARP7 is a member of the LARP family that contains four La domain-containing RNA binding proteins (LARP1, 4, 6, and 7) with distinct RNA target preferences (Bayfield et al., 2010). Among the four members, LARP7 is the only one that binds to 7SK snRNA and is involved in transcription elongation controlled by Pol II. The role of the LARP family proteins in human cancer is so far poorly known, and our study is the first demonstration that LARP7 functions as a potential suppressor of human breast cancer metastasis. This function of LARP7 is additionally supported by the observation that the Drosophila homolog of LARP7, multisex-combs (MXC), also acts as a tumor suppressor to inhibit cell growth (Remillieux-Leschelle et al., 2002). Moreover, the C-terminal region of LARP7, which is essential for the sequestration of P-TEFb in 7SK snRNP, is required for LARP7's suppression of breast cancer survival and is frequently deleted in human gastric cancer (Mori et al., 2002), suggesting that LARP7 may be a potential tumor suppressor in a broad range of human carcinomas. Finally, an inactivating mutation of LARP7 has been linked to a novel form of familial Primordial Dwarfism characterized by facial dysmorphism and intellectual disability (Alazami et al., 2012). In these patients, the 7SK snRNA is depleted due to the defective LARP7, indicating that LARP7 and 7SK snRNA, through their ability to control P-TEFb activity, also regulate embryonic development. Although it is formally possible that the LARP7 KD may also affect other yet-to-be-identified pathways in a P-TEFb-independent manner, our observation that both flavopiridol and siCDK9s efficiently rescued the increased cell migration of LARP7 KD cells lends a strong support to the notion that the observed effects of LARP7 KD on EMT and invasion are indeed mediated by P-TEFb activation.

A key observation of our study is that among the many EMT genes whose expression is altered by P-TEFb activation, the transcription factors Slug, FOXC2, ZEB2, and Twist1 are direct targets of P-TEFb and bound by the P-TEFb-containing SEC. These four proteins are well known EMT transcription factors that function as decisive early drivers of EMT. While FOXC2 orchestrates the mesenchymal component of the EMT program (Mani et al., 2007), Slug, ZEB2, and Twist1 repress E-cadherin expression, a fundamental event in EMT, as well as the expression of proteins of tight junctions, gap junctions, and desmosomes, contributing to the de-differentiated state of tumor cells and facilitating EMT (Peinado et al., 2007; Sanchez-Tillo et al., 2012). In addition, these proteins are also closely linked to breast cancer stem cell properties and implicated in resistance to drug and radio-therapies (Voulgari and Pintzas, 2009; Singh and Settleman, 2010; Hollier et al., 2013). Furthermore, these EMT early drivers also activate the expression of other EMT transcription factors. For example, Twist1 can induce Snail (Smit et al., 2009), and Slug can upregulate Twist2 and ZEB1 expression (Wels et al., 2011; Guo et al., 2012). Thus, by directly activating the expression of these EMT early drivers, P-TEFb is capable of orchestrating the EMT program to drive malignant progression. Since P-TEFb and its associated factors are expressed in most cell types, it is possible that the ability of P-TEFb to promote tumor progression may not be restricted to breast cancer but also true for other malignant tumors. Indeed, the active P-TEFb-Brd4 complex has been linked to AML and CML (Dawson et al., 2011; Mertz et al., 2011; Zuber et al., 2011; Herrmann et al., 2012; Winter et al., 2012), while multiple SEC components are direct targets of MLL-fusion proteins that are linked to various forms of leukemia (Lin et al., 2010; Yokoyama et al., 2010).

We have found that in the noninvasive breast cancer cell lines, P-TEFb released from the 7SK snRNP is mainly redistributed to the SEC. The expression levels of two SEC components AF9 and ELL2 are also markedly upregulated, allowing more SEC formation. Since ELL2 is another key catalytic component of SEC besides P-TEFb, its increase is likely to further enhance transcriptional elongation of target genes. We found that the Brd4-P-TEFb complex was also increased moderately upon LARP7 KD (data not shown), suggesting the possibility that Brd4 may also play a role in facilitating the P-TEFb-dependent breast cancer progression.

Given that P-TEFb is mostly known as a general transcription factor, our observations raise two interesting questions: Why are EMT transcription factors particularly sensitive to P-TEFb activation in these cells, and how is the SEC involved in this process? Our data that P-TEFb and SEC are associated with the genes encoding decisive EMT regulators but not non-responsive genes suggest that the direct target EMT genes may contain sequences that display high affinity binding to the SEC-P-TEFb complex. For example, these genes may contain the recently discovered Super-Enhancers that render them highly sensitive to activation by the Mediator and the SEC (Hnisz et al., 2013; Loven et al., 2013; Whyte et al., 2013). Interestingly, previous proteomic and biochemical analyses have identified the Mediator as a SEC binding partner through interaction between the Mediator subunit MED26 and the SEC components ELL-associated factor 1 (EAF1) and EAF2 (Takahashi et al., 2011), suggesting that the Mediator may recruit the SEC to target genes and the two complexes may act together to activate genes containing the Super-Enhancers. The SEC has also been linked to the elongating Pol II on chromatin template through interaction between the SEC components ENL/AF9 and the Pol II-associated factor 1 (PAF1) (He et al., 2011). Finally, SEC may be recruited to its target promoters through tissue-specific and sequence-specific DNA binding partners, such as the HIV Tat protein in HIV-infected cells, MLL-fusion proteins in leukemia cells (He and Zhou, 2011; Smith et al., 2011) or retinoic acid receptor in differentiating mouse embryonic stem cells (Lin et al., 2011). Besides the positive control exerted by P-TEFb and SEC, another possibility is that the EMT genes are strongly suppressed by negative transcription elongation factors DSIF and NELF, and thus are very sensitive to P-TEFb activation. Future studies will try to determine which of these mechanisms may be responsible for the heightened sensitivity of EMT transcription factors to the elongation control.

Taken together, our study has demonstrated that LARP7 functions as a potential tumor suppressor in human breast cancer by suppressing the activity of P-TEFb and inhibiting EMT, invasion, and metastasis. Our study also provides the first demonstration that the transcription elongation machinery and the network of P-TEFb complexes play critical roles in regulating tumor EMT, cancer stemness, and metastasis by directly controlling the expression of upstream EMT regulators. These findings will facilitate the development of new drugs that target CDK9 for anti-cancer therapy.

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Author details

1. Xiaodan Ji

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   XJ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Huasong Lu

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. School of Pharmaceutical Sciences, Xiamen University, Xiamen, China

   Contribution

   HL, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Qiang Zhou

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   QZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Kunxin Luo

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States

   Contribution

   KL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   kluo@berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

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