The Philip Hinds Lab
Cellular proliferation control in differentiation and cancer
The focus of my laboratory is on cellular mechanisms of proliferation control in progenitor cells and differentiation, and the dysregulation of these functions in cancer cells. We have two major areas of emphasis in this regard, and employ mouse models and engineered cell lines to produce preclinical models of tumorigenesis and developmental disorders. First, we are investigating the function of the retinoblastoma protein, (pRb), D-type cyclins, cdk4 and cdk6 in programs of cell cycle exit. Together, along with p16INK4a, a negative regulator of cdk4 and cdk6, these proteins regulate a critical decision making point in progression to S phase and thus DNA replication. In almost all human tumors, one of these proteins is lost or overexpressed, thus favoring proliferation and tumor growth. Although these proteins regulate S phase in response to genome integrity (DNA damage), we and others have shown that they are also critically involved in the permanent cell cycle withdrawal associated with differentiation and senescence, and we study these processes primarily in bone and breast cancer, as well as in cardiac disease.
Our second preclinical model of cancer focuses on the collaboration of the common melanoma oncogene BRAFV600E with individual members of the AKT kinase family. Murine genetic models and precisely engineered human cell lines are being used to identify pharmacologically targetable functions of each isoform to increase the spectrum of targeted and immunotherapeutic interventions in metastatic melanoma.
pRb and cdk6 in Bone Cell Differentiation and Senescence
We are presently studying the role of pRb in bone cell differentiation and senescence, with the aim of identifying specific mediators of the tumorigenic effects of pRb loss. Because loss of a tumor suppressor such as pRb cannot be simply restored as a therapeutic modality in cancer, identification of targetable, downstream alterations in pRb-null bone cancers may offer novel ways to prevent metastatic disease. To achieve this goal, we have constructed mouse models of pRb loss or cdk6 dysregulation in the bone, mimicking common events in human osteosarcoma. This has allowed us to begin careful studies of the phenotypic consequences of pRb pathway alteration in the bone as well as provide a source of genetically defined primary cells for culture-based studies of osteoblast differentiation. To precisely define the role of cdk6 in the bone, we have built mice bearing knockin alleles of hyperactive and “kinase dead” cdk6 (as well as null control). These animals are of great benefit as a source of primary cells for in vitro study, allow significant genetic experiments in crosses with existing animals prone to a variety of cancers, and are of lab-wide utility since cdk6 is likely to play roles in many of the processes studied in each of the above-mentioned areas. All of these studies have culminated in an overarching hypothesis that places the RB pathway at the center of cell fate decision-making steps in stem- and progenitor cells, and our major focus is now on understanding how this role of the RB pathway influences its role in cancer and development.
Figure 1. Rb loss increases the osteoblast/adipocyte progenitor, as shown by increased adipogenesis in mouse calvarial cells, bottom.
Cyclin D/cdk6 functions in epithelial, stromal and immune functions in cancer
In a separate albeit conceptually related project, we have produced "knock-in" alleles of cyclin D1 to test for novel roles for this protein in development and tumorigenesis. An important goal of these experiments is to clearly define the role of cyclin D1 in mammary tumorigenesis. To that end, we have crossed knockin animals with those prone to breast cancer to ask the important question of whether kinase activation is needed for tumorigenesis in the breast. Mice bearing a “kinase dead” allele of cyclin D1 fail to form breast tumors due to a paucity of a specific type of developmental progenitor cell in the affected mammary gland. Our ongoing work suggests that these cells are able to undergo ectopic proliferation in response to aberrant ErbB2 signaling and evade senescence, yet have vastly increased autophagy. This work has interesting implications for the use of cdk4/6 inhibitors to stimulate an autophagy-based synthetic lethality in certain breast tumor types, and possibly in other cyclin D1-driven tumors as well. Interestingly, our current work suggests that inhibition of the cyclin D partner kinase cdk6 impacts mammary epithelial hyperplasia and fibroblast activation, as well as T cell functions, implicating this kinase in a complex set of tumor and stromal cell interactions in nascent tumors.
Figure 2. A cyclin D1 mutant incapable of activating cdks blocks mammary tumor formation without impacting MMTV-ErbB2-driven proliferation in the mammary gland in vivo. Whole mounts and KI67 staining demonstrate ectopic epithelial cell proliferation in mice expressing MMTV-ErbB2 and inactive cyclin D1 (KE mutant; left panels). Right, top: preneoplastic lesions form in MMTV-ErbB2;KE/KE mice but show highly reactive stroma, indicating changes in the preneoplastic microenvironment. Right, bottom: despite increased epithelial proliferation, MMTV-ErbB2;KE/KE mice fail to form mammary tumors in contrast to WT controls.
Genetic interaction of AKT isoforms with BRAFV600E in metastatic melanoma
Finally, each of these areas of focus on development and tumorigenesis is complemented by our ongoing studies of a mouse model of BRAFV600E function in melanoma. This mouse faithfully recapitulates many aspects of human melanoma driven by this oncogene, the most common driver mutation in melanoma. These murine studies, as well as our parallel studies using engineered human melanoma cell lines, have revealed critical roles for individual members of the AKT family of signaling kinases in migration, invasion, cellular metabolism and metastasis in melanoma, reflecting significant progress toward our overarching goal of using genetics and preclinical models to identify novel targets appropriate for combinatorial approaches in melanoma therapy.
Figure 3. A. Phenotype of transgenic mice expressing high (470) or low (476) penetrance BRAFV600E oncogene. B. Survival curve of 476 mice crossed with knockout of tumor suppressor gene ARF. Asterisks indicate mice that developed spontaneous cutaneous melanoma. C. Engineered human cell lines were injected into nonimmune mouse tail vein, followed by doxycycline induction of anti-AKT shRNA and imaging as indicated. D. In vivo imaging of tail-vein injected mice reveals a significant delay in metastasis following isoform-specific knockdown of AKT in vivo.