Pancreatic cancer with Nest-in tendencies

In the previous issue of Cancer Biology & Therapy, Matsuda et al. present a role for Nestin in pancreatic ductal adenocarcinoma (PDAC) migration, invasion and metastasis.¹ Nestin, a class VI intermediate filament, has been implicated in cancer progression in a variety of tissues including the pancreas.^2–8 Nestin was originally identified as a neural stem cell marker and it is implicated in stem cell self-renewal.^9,10 However, the exact role of Nestin in cancer progression and the possible contribution of the stem cell in this context is unknown.

Although Nestin is an intermediate filament protein, its cellular functions are diversified beyond the structural (summarized in Table 1). Nestin is expressed during embryogenesis in actively proliferating precursor cells of the central nervous system and muscle.^9,11,12 Nestin is downregulated in differentiated cells but remains present in small populations of cells in adult tissues, and it is upregulated during tissue regeneration. These observations highlight a role for Nestin in cellular proliferation, and they also led to the hypothesis that Nestin is a progenitor/stem cell-specific protein. Nestin knockout mice are embryonic-lethal and have fewer neural stem cells in the developing neural tube.¹⁰ Neural stem cells derived from Nestin knock-out mice have decreased self-renewal capacity but do not show any differences in cellular proliferation or, interestingly, any effects on cytoskeleton integrity.¹⁰ Nestin has been identified in other tissues undergoing differentiation and/or regeneration including the testes, teeth, pancreas and kidney,^13–17 and it is also implicated in neo-vascularization in the hair follicle, skin and colon.^18–20 The wide variety of tissues in which Nestin is important suggests a conserved function in stemness or regeneration. However, Nestin can also contribute to cancer progression.

Table 1.

Supporting evidence for nestin involvement in various biofunctions

Innervation	Kawamoto, et al.24
Neo-vascularization	Amoh, et al.18 Aki, et al.9 Teranishi, et al.20 Gravdal, et al.21
Tissue regeneration	Kim, et al.15 Vansthertem, et al.17
Stem cell differentiation	Kachinsky, et al.11 Sejersen, et al.12 Frojdman, et al.13
Stem cell self-renewal	Park, et al.10
Cell migration, invasion and metastasis	Matsuda, et al.1 Chen, et al.3 Kleeberger, et al.4 Florenes, et al.22
Tumor progression	Krupkova, et al.8 Yang, et al.2 Li, et al.5 Lim, et al.6 Piras, et al.7

Cancer cell growth mirrors the proliferative aspects of both tissue regeneration and stem cell self-renewal. The cancer stem cell hypothesis proposes that a transformed progenitor/stem cell, with its infinite regenerative capacity, can give rise to a tumor. Furthermore, the cancer stem cell must be targeted therapeutically in order for the tumor to ultimately disappear. Nestin expression has been correlated with tumor severity in neuroblastomas, gliomas, melanomas and prostate cancers.^2,4,7 Given its expression patterns in stem cells, regenerating tissues and neovascularization, its correlation with tumor severity aligns with the cancer stem cell hypothesis. However, its direct roles in tumor progression are unknown.

There are other hypotheses for how Nestin may contribute to tumor progression in addition to its potential role in maintaining (cancer) stem cell self-renewal. Tumors require blood vessel infiltration or angiogenesis in order to survive, and Nestin is expressed in the endothelial cells that generate the vasculature.^20,21 However, this data is correlative, and the necessity for and mechanism of action of Nestin in tumor angiogenesis has yet to be tested. The most-studied hypothesis for how Nestin contributes to tumor progression is that it impacts cell motility. Nestin correlates with tumor severity, and aggressive tumors are characterized by higher levels of invasion, migration and metastasis.

Kleeberger et al. reported that while Nestin did not affect prostate cancer cell viability or proliferation, it was necessary for cell motility and invasion in vitro.⁴ Furthermore, Nestin appeared to mediate prostate cancer cell migration and metastasis in xenograft experiments in vivo. These experiments emphasized a role for Nestin in cell motility rather than proliferation. Nestin is also implicated in melanoma cell invasion, although its contribution has not been challenged directly.²² It is also a potential marker for circulating melanoma cells, which may serve a prognostic purpose. 23 Kawamoto et al. previously showed that Nestin was expressed in ∼30% of pancreatic cancer patient samples and did not correlate with prognosis or survival.²⁴ However, they observed Nestin expression at sites of peripheral nerve fibers and adjacent stromal tissue, suggesting that Nestin may contribute to a tumor micro-environment that promotes invasion. As in the examples above, much of the data surrounding the role of Nestin in cancer progression is correlative, and mechanistic studies are needed.

The study by Matsuda et al. focuses on the role of Nestin in pancreatic ductal adenocarcinoma (PDAC) invasion.¹ Pancreatic cancer is the fourth-deadliest cancer in American men and it has a 5-year survival rate of only 5–6%.²⁵ PDAC is the most common presentation of pancreatic cancer, accounting for ~85% of cases.²⁶ It is a highly invasive cancer, and it rapidly metastasizes to the lymphatic system, spleen, liver and lungs at very early stages in tumor development.²⁶ It is commonly treated with surgery and/or gemcitabine, sometimes in combination with oxaliplatin, but response to chemotherapy is very poor and new regimens need to be developed that impede the rapid tumor cell dissemination that is characteristic of this tumor.

Because Nestin is expressed in ∼30% of PDAC cases and is implicated in cellular invasion and motility, Matsuda et al. hypothesized that Nestin contributes to PDAC invasion, migration and metastasis.¹ They transfected two PDAC cell lines (PANC-1 and PK-45H) with scrambled control or Nestin shRNA. Nestin knockdown led to increased cell-cell contact and elevated levels of F-actin at the cell periphery. Nestin knockdown had no effect on PDAC growth potential in vitro or subcutaneously in vivo. However, Nestin contributed to PDAC cell migration and invasion in vitro according to scratch assays, Boyden chamber assays and modified Boyden chamber assays with matrigel.

To investigate a possible mechanism behind Nestin-mediated migration and invasion, the authors performed a PCR array for genes associated with metastasis. The most highly upregulated gene in Nestin knockdown cells relative to control cells was E-cadherin. Immunofluorescence experiments revealed that E-cadherin was localized to cell-cell junctions at the cell membrane of Nestin knockdown PDAC cells. E-cadherin is best characterized as a mediator of epithelial-to-mesenchymal transition (EMT), which is often a prerequisite for cellular invasion and migration. During EMT initiation, the transcription factors Twist, Slug and Snail suppress E-cadherin to allow decreased cell-cell adhesion. Twist was undetectable in the PDAC cells, but Slug mRNA was increased in Nestin knockdown PANC-1 cells relative to control cells and Snail mRNA was decreased in PK-45H cells. Slug levels did not change in the Nestin knockdown PK-45H cells compared to the control, while Snail levels did not change in the Nestin knockdown PANC-1 cells. The disparity in expression of these three transcription factors suggests that E-cadherin is regulated in myriad ways, and the mechanistic connection between Nestin and E-cadherin remains unknown. Finally, the authors observed decreased liver metastases of Nestin knockdown PANC-1 cells compared to control six weeks after injecting the cells into the spleens of mice. Of note, the proliferative capacities of the metastatic outgrowths were the same in the Nestin knockdown cells and the control cells, according to Ki-67 immunostaining. Together, this report shows that Nestin contributes to PDAC cell invasion, migration and metastatic capability, but not to cellular proliferation. Furthermore, Nestin expression negatively correlates with E-cadherin expression, and an absence of E-cadherin may promote EMT and the invasive phenotype.

This report corroborates those that present a role for Nestin in cell invasion and migration but not proliferation, which is of particular interest for studying a highly metastatic cancer such as PDAC. It proposes a relationship between Nestin, an intermediate filament and E-cadherin, an adhesion protein implicated in EMT. Nestin expression was also found to be inversely correlated with E-cadherin expression in aggressive anaplastic thyroid carcinoma cells.²⁷ Interestingly, E-cadherin was recently implicated as a moderator of neural stem cell self-renewal, but in that setting E-cadherin expression was positively-correlated with Nestin-positive neural stem cells.²⁸ The mechanistic functions of Nestin are likely complex, and they are sure to differ with cellular context. For all of the hypotheses and initial evidence for the multiple ways that Nestin may impact cancer progression, mechanistic proof is still needed and Matsuda et al. are up for the challenge.

Commentary to: Matsuda Y, Naito Z, Kawahara K, Nakazawa N, Korc M, Ishiwata T. Nestin is a novel target for suppressing pancratic cancer cell migration, invasion and metastasis. Cancer Biol Ther. 2011;11:512–523.