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β4 Integrin signaling induces expansion of prostate tumor progenitors, "Beyond the Abstract," by Toshiaki Yoshioka, Javier Otero, and Filippo G. Giancotti

BERKELEY, CA (UroToday.com) - Increasing evidence suggests that oncogenic mutations exert their action by transforming normal stem cells or their immediate progeny, the transit-amplifying cells, into neoplastic progenitor cells, thereby spurring the development of hierarchically organized cancers, which consist of cancer stem cells and their aberrantly differentiated progeny.[1, 2, 3] The cancer stem cells are operationally defined by their ability to undergo self-renewal in vitro and to initiate secondary tumors upon xenotransplantation in mice. Moreover, there is evidence suggesting that these cells are largely dormant within outgrowing tumors, i.e., they are not proliferating or are doing so very slowly, and, hence, they are relatively resistant to both chemotherapy and oncogene-targeted therapy. However, therapy-induced eradication of the bulk tumor mass can induce their recruitment into the cell cycle, driving the relapses observed in the clinic.[4] In spite of considerable effort, the contextual cues that regulate normal stem cells and their neoplastic counterpart are poorly understood.

Prostatic glands are composed of a continuous layer of columnar secretory cells resting on a basal layer comprising basal cells and scattered neuroendocrine cells, separated by a basement membrane.[5] Prospective identification and lineage-tracing experiments have led to the identification of potential normal stem cells in both the basal and the luminal compartments of the mouse.[6, 7] Since human prostate cancers are characterized by a loss of normal basal cells and by an expansion of cells that morphologically and biochemically resemble luminal cells, it has been hypothesized that these tumors arise from neoplastic conversion of a luminal progenitor.[8, 9] In agreement with this hypothesis, lineage-tracing experiments have suggested that the luminal layer of the mouse prostate contains Nkx3-1–positive bi-potential progenitors, which can be converted into neoplastic cells by inactivation of the tumor suppressor Pten.[7] Basal cells are seemingly resistant to direct transformation, although loss of Pten induces them to differentiate into transformation-competent luminal cells.[10] In contrast, the luminal compartment of the human prostate is refractory to transformation in vitro by simultaneous introduction of activated Akt, ERG, and AR, whereas the basal cells contain bi-potential progenitors that can be transformed by this combination of oncogenes.[11, 12] The signaling pathways that govern the expansion of prostate tumor progenitor cells are incompletely understood. Adult stem cells undergo self-renewal and differentiation in response to contextual cues originating from the specialized microenvironment (“niche”) in which they reside.[13] Because of its ability to support cell adhesion and signaling by binding to integrins and its presence in many stem cell niches, the basement membrane appears to be well suited to regulate stem cell behavior.[14]

The α6β4 integrin (referred to as β4 integrin because β4 pairs only with α6) binds to laminin-5, a major component of the basement membrane of the prostate,[15] and is characterized by a signaling mechanism that is unique among integrins.[16, 17] Following β4 binding to laminin-5, the distal segment of the large cytoplasmic domain of β4 (referred to as the signaling domain) is phosphorylated by a Src family kinase and recruits the signaling adaptor protein Shc, causing activation of Ras and PI-3K.[18, 19, 20, 21] Furthermore, the β4 integrin can combine with multiple receptor tyrosine kinases (RTKs) to promote intracellular signaling.[22, 23, 24, 25] Interestingly, in vitro studies have indicated that oncogenic forms of c-Met can induce invasive signaling through phosphorylation of the β4 cytoplasmic domain.[23] Moreover, genetic analysis has revealed that the β4 integrin promotes mammary tumorigenesis by combining with ErbB2 and amplifying its signaling capacity.[25] However, it is not clear whether β4 can exert a pro-neoplastic function in cancers that do not carry activating mutations or genomic amplifications of its partner RTKs, such as prostate carcinomas.[26]

A prior pathological study of low Gleason grade prostate cancer suggested that the β4 integrin is downregulated concomitant with loss of normal basal cells.[27] However, by using Cancer Outlier Profile Analysis (COPA) we identified the ITGB4 gene, encoding β4, as markedly overexpressed in a subset of tumor samples in 11 out of 16 available DNA microarray data sets. Interestingly, high-level expression of ITGB4 was associated with high Gleason grade in primary tumors as well as with androgen-independent metastases to bone. Furthermore, immunohistochemistry on paraffin-embedded sections indicated that the neoplastic cells in virtually all PIN lesions and a significant fraction of invasive prostate cancers expressed the β4 integrin, often in combination with c-Met and ErbB2.

Motivated by these observations we examined if β4 signaling promotes prostate tumorigenesis by introducing a targeted deletion of the β4 signaling domain into PB-TAg mice, which are engineered to develop prostate cancer. We found that loss of β4 signaling inhibits tumor growth and delays progression to metastasis in these mice. Interestingly, deletion of the β4 signaling domain impaired the ability of prostate tumor cells to form tumor spheres in vitro (an assay used to measure the capacity of tumor progenitor cells to undergo self-renewal in vitro) and to proliferate in vivo. In addition, tissue recombination experiments revealed that prostate epithelial-specific deletion of the β4 signaling domain suppresses oncogenesis driven by loss of Pten, a common lesion in human prostate cancer. These findings suggest that integrin β4 signaling sustains both self-renewal and rapid proliferation of tumor progenitors in vivo. Furthermore, since the expression of β4 is enriched in the fraction of tumor cells attached to the basement membrane, our results support the model that prostate tumors contain a basal compartment consisting of putative stem cells able to undergo self-renewal, as well as a compartment of transient-amplifying cells undergoing rapid proliferation.

The RTKs ErbB2 and c-Met appear to be activated in a fraction of human prostate cancers, either because of overexpression or in response to a paracrine signaling loop.[28, 29, 30] Our results indicate that inactivation of β4 signaling decreases activation of ErbB2 and c-Met, impairing downstream signaling to Akt and ERK in both cancer stem cells and transit-amplifying cells. Moreover, we found that the EGF-R/ErbB2 inhibitor lapatinib and the c-Met inhibitor crizotinib cooperate to suppress cancer growth and to induce apoptosis in the DU145 xenograft model of prostate cancer, whereas each of the two agents exerted a modest effect when used alone. These results raise the possibility that a combination of these or similar agents inhibiting ErbB2 and c-Met may display therapeutic efficacy in castration-resistant prostate cancer, or in the androgen-dependent phase of the disease, in combination with drugs targeting the AR. For example, a rational approach would be target the bulk of AR-dependent prostate cancer cells with anti-androgen therapy and strike the residual cancer stem cells with agents targeting ErbB2 and c-Met.

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Written by:
Toshiaki Yoshioka,a, b,* Javier Otero,a and Filippo G. Giancottia as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

aCell Biology Program, Sloan-Kettering Institute for Cancer Research and Metastasis Research Center, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, USA.
bDepartments of Molecular Pathology and Tumor Pathology, Akita University Graduate School of Medicine, Akita, Japan.

*Correspondence:

β4 Integrin signaling induces expansion of prostate tumor progenitors - Abstract

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