Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy

Identification and Targeting of Stem Cell Signals in Cancer

It is common to hear scientists equate targeting cancer to playing whack-a-mole: hitting one “mole” (or pro-tumorigenic signal) will lead to the emergence of another mole, then another, and so on. This analogy is far too simple and disregards the elegant evolution observed in cancer. In this respect, I propose that cancer is more comparable to a pin art board. At one moment the pins are in the shape of a hand. If asked to describe the pin art board, all would agree that fingers and a palm are critical components. In the next moment the hand is replaced with a face defined by eyes and a mouth. Although these two snapshots seem quite different, there are underlying mechanics that can explain the fluidity that permits the hand to morph to a face, and those mechanics are shared across all pin art boards. In a similar way, cancer is quite fluid. At one moment the tumor is addicted to a signaling axis that can be therapeutically targeted. Upon blockade of that addiction, the tumor is able to elegantly morph into a new state with an entirely new set of defining features. This “new tumor state” is not a random “mole” that sporadically pops up in an unpredictable manner; rather, the tumor can predictably hijack mechanisms that permits fluid state changes. Instead of focusing on the mole, or better yet the shape of the pin art board, here we try to understand the mechanics and drivers of what defines the observed tumor phenotype. What allows tumor cells to overcome toxic insults, and adapt to novel and harsh environments? To this end, tumor initiation, therapy resistance, and metastasis are highly dependent on the ability for cells to adapt and survive, and thus represent processes that select for the most adaptable, aggressive cell type. The following thesis aimed to identify and characterize tumor cells with the intrinsic capacity to thrive during those processes. Particular focus was given to tumor cells with stem cell properties, as these are characteristically able to respond to stresses and regenerate tumors accordingly. Using pancreatic cancer as a model system, we identified a population of tumor cells, functionally marked by the stem cell fate determinant Musashi, with cancer stem cell characteristics: Musashi+ cells were enriched for tumor propagating capacity, highly resistant to cytotoxic therapies, and preferentially survived in circulation. Further, we carried out unbiased screens to understand the landscape that defined these pancreatic cancer stem cells. This led to the discovery of targetable signals required for maintenance of a stem cell state, and thus represents an exciting new therapeutic approach designed to collapse mechanisms that promote an adaptable, aggressive cell phenotype. Finally, I highlight the power of using intravital imaging to track tumor cell behaviors in vivo, and apply this tool to better understand intrinsic and extrinsic dependencies

Identification and Targeting of Stem Cell Signals in Cancer

It is common to hear scientists equate targeting cancer to playing whack-a-mole: hitting one “mole” (or pro-tumorigenic signal) will lead to the emergence of another mole, then another, and so on. This analogy is far too simple and disregards the elegant evolution observed in cancer. In this respect, I propose that cancer is more comparable to a pin art board. At one moment the pins are in the shape of a hand. If asked to describe the pin art board, all would agree that fingers and a palm are critical components. In the next moment the hand is replaced with a face defined by eyes and a mouth. Although these two snapshots seem quite different, there are underlying mechanics that can explain the fluidity that permits the hand to morph to a face, and those mechanics are shared across all pin art boards. In a similar way, cancer is quite fluid. At one moment the tumor is addicted to a signaling axis that can be therapeutically targeted. Upon blockade of that addiction, the tumor is able to elegantly morph into a new state with an entirely new set of defining features. This “new tumor state” is not a random “mole” that sporadically pops up in an unpredictable manner; rather, the tumor can predictably hijack mechanisms that permits fluid state changes. Instead of focusing on the mole, or better yet the shape of the pin art board, here we try to understand the mechanics and drivers of what defines the observed tumor phenotype. What allows tumor cells to overcome toxic insults, and adapt to novel and harsh environments? To this end, tumor initiation, therapy resistance, and metastasis are highly dependent on the ability for cells to adapt and survive, and thus represent processes that select for the most adaptable, aggressive cell type. The following thesis aimed to identify and characterize tumor cells with the intrinsic capacity to thrive during those processes. Particular focus was given to tumor cells with stem cell properties, as these are characteristically able to respond to stresses and regenerate tumors accordingly. Using pancreatic cancer as a model system, we identified a population of tumor cells, functionally marked by the stem cell fate determinant Musashi, with cancer stem cell characteristics: Musashi+ cells were enriched for tumor propagating capacity, highly resistant to cytotoxic therapies, and preferentially survived in circulation. Further, we carried out unbiased screens to understand the landscape that defined these pancreatic cancer stem cells. This led to the discovery of targetable signals required for maintenance of a stem cell state, and thus represents an exciting new therapeutic approach designed to collapse mechanisms that promote an adaptable, aggressive cell phenotype. Finally, I highlight the power of using intravital imaging to track tumor cell behaviors in vivo, and apply this tool to better understand intrinsic and extrinsic dependencies

The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in <i>Drosophila</i>

<div><p>Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (<i>stai</i>) gene in the maintenance of axonal microtubules and regulation of axonal transport in <i>Drosophila</i>. The <i>stai</i> gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of <i>stai</i> function in <i>Drosophila</i>, which has a single <i>stai</i> gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the <i>stai</i> gene family. This has allowed us to identify an essential function for <i>stai</i> in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of <i>stai</i> mutant <i>Drosophila</i>, we also observe additional neurological phenotypes associated with loss of <i>stai</i> function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in <i>stai</i> deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of <i>Drosophila</i> mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that <i>stai</i> has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.</p> </div

Loss of <i>stai</i> Function Results in Defects in Fast Axonal Transport.

<p>Mutations in <i>stai</i> result in phenotypes consistent with axonal transport defects. (<b>A</b>) Wildtype third instar larvae maintain a flat body posture when crawling. In contrast, homozygous <i>stai</i>^<i>B200</i> larvae (<b>B</b>) and <i>stai</i>^<i>rdtp</i> larvae (<b>C</b>) exhibit a posterior paralysis “tail flip” phenotype common for <i>Drosophila</i> mutants defective in axonal transport (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068324#pone.0068324.s002" target="_blank">Movies S1</a>-S3). The tail-flip phenotype is also observed in heterozygous <i>stai</i>^<i>B200</i>/<i>stai</i>^<i>rdtp</i> larvae (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068324#pone.0068324.s005" target="_blank">Movie S4</a>). Confocal micrographs of sections of segmental nerve from wildtype (<b>D</b>), <i>stai</i>^<i>B200</i><i>/+</i> heterozygous (<b>E</b>) and <i>stai</i>^<i>B200</i> homozygous (<b>F</b>) third instar larvae stained with antibody against synaptic vesicle protein cysteine string protein (CSP). (<b>D</b>) The axons of wild type animals showed faint, punctate staining for CSP uniformly throughout the axons within the compound nerve bundle. (<b>E</b>) Although heterozygous <i>stai</i>^<i>B200</i> larvae showed no obvious sluggishness or tail-flip phenotype when they crawled, mild accumulations of CSP were observed in some segmental nerve axons. (<b>F</b>) In contrast, homozygous mutant <i>stai</i>^<i>B200</i> larvae exhibit abundant accumulations of CSP throughout the segmental nerve, confirming a disruption in axonal transport. (<b>G</b>) When <i>stai</i>^<i>B200</i> is placed in heterozygous combination with <i>Df</i>(<i>2L</i>) <i>Exel6015</i>, a chromosomal deletion that spans the <i>stai</i> locus, there is an enhancement in the abundance of axonal accumulation of CSP. (<b>H</b>) The severity of axonal transport defects was quantified by averaging the number of accumulations of CSP immunopositive anterograde and retrograde membranous axonal cargos observed in the segmental nerve axons of <i>stai</i> deficient third instar larvae. To determine if there was an anterior-to-posterior gradient in the severity of axonal transport defects, regions of segmental nerve axons were analyzed as they passed through abdominal segment A2 (light grey bars) and abdominal segment A4 (dark grey bars). Data is also presented as total number of accumulations observed in both abdominal regions (black bars). The only significant regional difference observed in the abundance of axonal clogs in segmental nerve axons between abdominal segment A2 and A4 was in the axons examined from <i>stai</i>^<i>B200</i><i>/Df</i>(<i>2L</i>) <i>Exel6015 larvae</i> (<i>p<0.0001</i>). Wild type larvae exhibited 0.57 ± 1.21 axonal clogs/50 µm segmental nerve axon in all axons examined. Homozygous <i>stai</i>^<i>B200</i> larvae averaged 13.55 ± 5.56 axonal clogs/50 µm segmental nerve axon (p<0.0001). The average number of axonal clogs observed in the segmental nerve axons of <i>stai</i>^<i>B200</i><i>/Df</i>(<i>2L</i>) <i>Exel6015</i> larvae was 15.10 ± 6.07 axonal clogs/50 µm segmental nerve axon. In panels A-C, the scale bar = 1mm. In panels D-G, the scale bar = 10 µm.</p

Loss of <i>stai</i> Function Reduces Levels of Stabilized Axonal Microtubules.

<p>(<b>A</b>–<b>F</b>) Confocal micrographs of segmental nerve axons from third instar larvae immunostained with antibody against acetylated α-tubulin (mAb 611B1). Axons from (<b>A</b>) wildtype, (<b>B</b>) <i>stai</i>^<i>B200</i> and (<b>C</b>) <i>stai</i>^<i>B200</i><i>/Df</i>(<i>2L</i>) <i>Exel6015</i> larvae. (<b>D</b>) Precise excision of the <i>piggyBac</i> element <i>PBac</i>{<i>5HPw⁺</i>}^<i>B200</i> reverts the reduction in acetylated α-tubulin back to wild type levels. The reduction in acetylated α-tubulin staining intensity observed in <i>stai</i> deficient segmental nerve axons is significantly restored with the ubiquitous ectopic expression of <i>Drosophila staiB2</i> (<b>E</b>) or human STMN1 (<b>F</b>). (<b>G</b>) Quantification of average pixel intensity of acetylated α-tubulin immunostained axons of third instar larval segmental nerves. Results are presented in arbitrary units (a.u.) of fluorescence intensity (mean ± S.D.). In panels A-F the scale bar = 10 µm.</p

Identification of Mutations in the <i>stathmin</i> (<i>stai</i>) Locus.

<p>(<b>A</b>) Genomic structure of the <i>Drosophila</i><i>stai</i> locus and the positions of the mutagenic copia retrotransposon <i>stai</i>^<i>rdtp</i> and the <i>piggyBac</i> element <i>stai</i>^<i>B200</i>. Exons are boxed, noncoding portions of exons are white, exons 3, 4, 5, shared by all stai proteins, are light grey (after Lachkar et al, 2010). Transcripts encoding <i>staiA</i> isoforms include exons 1 and 2 with alternative splicing that either includes (<i>staiA1</i>) or excludes (<i>staiA2</i>) exon 6. Transcripts encoding <i>staiB</i> isoforms include exons 1’and 2’ with alternative splicing that either includes (<i>staiB1</i>) or excludes (<i>staiB2</i>) exon 6. The copia retrotransposon <i>stai^rdtp</i> is inserted in the open reading frame of the <i>stai</i> gene in exon 1', ten base pairs downstream of the translational start site used to produce nervous system enriched <i>staiB</i> encoding transcripts. The <i>piggyBac</i> element <i>PBac</i>{<i>5HPw⁺</i>}<i>stai^B200</i> is inserted in the 2.8 kb intron separating exons 1’ and 2’, 1.3kb downstream of the splice junction of exon 1’. (<b>B</b>) The copia insertion in the <i>stai</i> gene was identified by PCR amplification across the open reading frame of <i>stai</i> exon 1’ that resulted in an unexpectedly large 5.6 kb product from genomic DNA isolated from <i>stai</i>^<i>rdtp</i> homozygous larvae. The black arrowheads in Figure 1A represent the relative position of PCR primers used. (<b>C</b>) qRT-PCR of <i>staiA</i> and <i>staiB</i> mRNA derived from third instar larvae is shown. The expression of <i>staiA</i> and <i>staiB</i> is significantly reduced in all <i>stai</i> mutant genotypes analyzed compared to wild type expression levels (P<0.01). The red and blue arrowheads in Figure 1A represent the relative position of primers used for qRT-PCR of <i>staiA</i> and <i>staiB</i> message. Results are normalized against the expression of the <i>GAPDH</i> gene product and are presented as mean ± SD.</p