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Despite early benefits seen in cancer patients treated with anti-VEGF pathway targeted drugs, the clinical benefits obtained in terms of progression-free or overall survival have been more modest than expected. This is, at least in part, due to antiangiogenic drug resistance mechanisms that involve pathways mediated largely by the tumor, whether intrinsic or acquired in response to therapy, or by the host, which is either responding directly to therapy or indirectly to tumoral cues. The focus of this review is to distinguish, where possible, between such host and tumor-mediated pathways of resistance and discuss key challenges facing the preclinical/clinical development of antiangiogenic agents, including potential differences in drug efficacies when treating primary tumors or various stages of metastatic disease.
The concept of targeting the tumor’s vasculature to block its growth has been validated clinically as an anti-cancer strategy with the approval of three targeted drugs that disrupt the vascular endothelial growth factor (VEGF)/VEGF receptor pathway 1. However, despite encouraging signs from some early preclinical studies that prolonged benefits would be seen in cancer patients, recent findings from the laboratory and clinic have uncovered several limitations to antiangiogenic therapy, posing future challenges for their expanding use. Currently approved antiangiogenic drugs include bevacizumab, the humanized monoclonal antibody to VEGF, as well as small molecule receptor tyrosine kinase inhibitors (RTKIs), such as sorafenib and sunitinib, which target VEGF and platelet-derived growth factor (PDGF) receptors (among a number of others). The VEGF RTKIs (approved thus far as single agents) and bevacizumab (approved for use only in combination with cytotoxic chemotherapy) can lead to disease stabilization and longer periods of progression free survival (PFS) or overall survival (OS) in many patients with metastatic disease, including colorectal carcinoma (CRC), metastatic breast carcinoma (MBC), non-small cell lung carcinomas (NSCLC), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), gastrointestinal stromal tumors (GIST), and perhaps (though this has yet to be proven) in glioblastomas (GBM) (reviewed in 2). But tumors eventually become non-responsive, or do not respond at all — despite the presence of VEGF and VEGFR-2 - and PFS or OS in patients receiving antiangiogenic therapy has translated into benefits measured only in months, in most cases 3. Furthermore, in certain instances, increases in response rate and PFS does not always translate into increased OS for patients, as observed after bevacizumab treatment in RCC (as a single agent)4 or in MBC (in combination with a taxane chemotherapy) 5. It also remains unclear what role drug combinations play in the efficacy of VEGF pathway targeting (antiangiogenic) inhibitors and why, at least to date, bevacizumab has proved largely ineffective as a single agent while VEGF RTKIs, with one recent exception 6, have repeatedly failed in randomized phase III trials when used in combination with chemotherapy 7.
Thus there is a growing interest in understanding the mechanisms of resistance, whether intrinsic or acquired, after exposure to antiangiogenic drug treatment. Early indications are that these mechanisms may be highly diverse, perhaps in part due to the primary mode of action of such drugs, e.g. blocking ‘host’ tumor-supporting processes rather than blocking tumor growth directly. It is possible that resistance to antiangiogenic therapy may extend beyond classical drug resistance seen with traditional cytotoxic chemotherapy and radiation, or even molecular ‘tumor’ targeted therapy, which include rapid mutability and adaptability inherent to the tumor cells’ genetic instability (see review 8). Indeed an emerging question is whether the theoretical advantages of disrupting ‘host’ angiogenic processes, may be countered by significant disadvantages, including host-mediated resistance mechanisms involving the vascular microenvironment (perhaps largely independent of the tumor) as well as an altogether more disquieting possibility, namely, that antiangiogenic resistance may, in some instances, eventually increase or induce the invasive and metastatic potential of tumors as a result of therapy.
The focus of this review is to discuss two interrelated pathways. The first includes main pathways of resistance to antiangiogenic therapy, differentiating between those meditated by either the tumor itself or by the host (or both). The second pathway looks at disease progression from a localized primary tumor to established metastatic disease. It may be critical to consider both pathways simultaneously to understand and overcome some of these challenges facing antiangiogenic therapy, including mechanisms of drug resistance and how they may play a significant role in influencing tumor growth, for better or worse, at various stages of disease (Figure 1).
The initial non-response seen in a subset of patients receiving VEGF pathway-targeted therapy imply that various tumor cells possess certain intrinsic properties which could allow for immediate resistance upon treatment initiation. These could be dependent on a multitude of factors, such as patient treatment history, stage of disease, genetic factors, as well as inherent tolerability to hypovascular environments — something observed in certain cancer types (recently reviewed in 8). Some mechanisms of resistance thought to be mediated largely by the tumor include co-option of established vessels 9 and pre-existing expression by the tumor of multiple alternative proangiogenic pathways (i.e., PDGFs, PlGFs, FGFs) 10 — which could compensate immediately for the loss of VEGF signalling. Additional compensatory mechanisms may also be acquired by the tumor as a response to elevated tumor hypoxia induced by blockade of VEGF signalling and include the upregulation of alternate proangiogenic mediators, such as bFGF and SDF1α, that could allow for persistent neovascularisation despite continued anti-VEGF therapy 11,12. It is also evident that therapy-induced hypoxia plays a critical role in facilitating the selection of tumor cells which are able to tolerate, and perhaps even thrive, in low oxygen environments 13,14 (recently reviewed in 15).
While resistance to VEGF pathway targeted therapy may be mediated in large part by these intrinsic or acquired characteristics of the tumor cells, it is increasing clear that mechanisms involving the tumor microenvironment -- either directly (in response to drug action) or indirectly (in response to cues from the tumor) — can also be involved in mediating eventual tumor relapse and regrowth. For example, stromal cells such as tumor associated fibroblasts (TAFs) can upregulate PDGF-C in response to VEGF inhibition 16. Pericytes could also play a role by retaining vascular function following endothelial cell (EC) disruption 17, regulating EC proliferation 8,18, and/or providing a scaffold (along with remaining basement membrane associated cells) for rapid revascularization after cessation of therapy 19. Moreover, various types of proangiogenic bone marrow-derived cells (BMDCs) may home to the tumor microenvironment and mediate resistance to VEGF pathway blockade via the production of the aforementioned compensatory proangiogenic factors 3. Examples include circulating cells such as Gr1+CD11b+ myeloid suppressor-type cells 20 via Bv8 (prokineticin) and G-CSF-dependent mechanisms 21, TIE2 expressing monocytes via upregulation of angiopoeiten-2 22, and tumor-associated macrophages (also via upregulation of Bv8) 23, and there are likely others 24. Taken together, these tumor- and host-mediated mechanisms, either alone or together in concert, may diminish response to antiangiogenic agents despite continued therapy. But what are the phenotypic characteristics of tumors that progress after initial benefit with antiangiogenic therapy?
The progression of a locally growing primary tumor to the growth of distant metastases involves a number of steps, including a loss of cellular adhesion; augmented motility and invasion capabilities; intravasation into the bloodstream; honing and survival; extravasation and seeding of micrometastases; and finally colonization and growth in a new distant site 25. Because of the integral role of the vasculature in this process, one obvious theoretical advantage of antiangiogenic therapy would be that some of these steps may be compromised, particularly in primary tumors (e.g. via the destruction of the immature vasculature to prevent/suppress intravasation), as well as in distant sites (e.g., the prevention of avascular metastases which require angiogenesis for continued growth) To date, extensive preclinical and clinical studies using VEGF pathway-targeting drugs have indeed been shown to stop or slow the growth localized primary tumors or established metastatic disease but it remains largely unknown how effective antiangiogenic therapy is at blocking earlier stages of metastatic disease. Clues that antiangiogenic agents may not sufficiently suppress metastasis in many cases and, even more provocatively, possibly select for more invasive and metastatic tumor phenotypes, have recently emerged. For example, in preclinical tumor models of GBM where VEGF or HIF1α was genetically or therapeutically blocked 9,26,27, initially tumors shrank but elevated hypoxia in the tumor microenvironment eventually caused or facilitated recurrent tumor growth in existing and adjacent sites. Moreover, Paez-Ribes et al. recently showed that therapy with anti-VEGFR-antibodies or various VEGF RTKIs in genetically engineered RIP1-Tag2 pancreatic islet cell tumors and in orthotopically transplanted GBMs eventually resulted in tumors capable of increased invasion/intravasation and metastasis in distant organs (such as the liver) 28. Thus, in response to hypoxia induced by anti-VEGF pathway targeted therapy, tumors may acquire adaptive/evasive behaviour. Interestingly, tumor-independent (host-mediated) pathways of resistance to angiogenesis inhibition may also facilitate tumor growth and metastasis in certain instances. For example, following short-term (7 day) treatment with various VEGF RTKIs to mice prior to intravenous inoculation of human tumor cells, or immediately after removal of a primary tumor, accelerated metastasis could be observed concomitant with decreased survival 29.
But how can this be explained? Many possible mechanisms could be involved. For example, therapy-induced increases in tumor hypoxia and HIF1α expression following VEGF-pathway inhibition can lead to i) increased c-met expression 30,31 or IL6 expression 32, ii) activation/upregulation of various matrix metalloprotienases 33, iii) mobilization of BMDCs 34, iv) instigation of tumor epithelial-to-mesenchymal (EMT) transition 35 - all of which could increase invasive/metastatic potential in a ‘tumor-mediated’ manner. Tumor-independent (‘host-mediated’) mechanisms could contribute as well, for example, via aforementioned therapy-induced upregulation of various proangiogenic molecules — many of which may increase the invasive/metastatic potential of cancer cells. For instance, it is now well documented that increases in VEGF and PlGF, and decreases in sVEGFR-2, can be observed in the plasma of patients receiving VEGF RTKIs (including sorafenib, sunitinib, and many others) such that it can be considered a ‘class effect’ for these agents (see Supplementary Table 1 in 36). Indeed this is a major reason why these proteins are currently being evaluated for use as potential surrogate biomarkers for tumor response 37,38. However many of these changes could derive, in large part, from a systemic host-mediated response to treatment rather than (or in addition to) from the tumor itself. This possibility was raised by recent experiments from our laboratory which showed that dose-dependent, reversible, elevations in VEGF/PlGF (and decreases in sVEGFR-2) could be recapitulated in healthy tumor-free mice treated with VEGF RTKIs and could include many ‘off-target’ molecules such as osteopontin, G-CSF, and SDF1α 12,36. Given that many of these circulating proangiogenic cytokines, chemokines, and growth factors have been implicated in promoting angiogenesis and/or metastasis 39-43, it is theoretically possible that they may assist in the aforementioned rebound revascularization 19 and/or increased extravasive potential for circulating tumor cells. Such induced systemic host responses to antiangiogenic drugs could facilitate an enhanced ‘pre-metastatic niche’ precipitated by mechanisms largely independent of the tumor 29,44. These include, i) BMDC mobilization, such as the recruitment of circulating VEGFR-1+ bone marrow cells to distant organ sites 45,46, ii) pericyte dysfunction, which may in turn make vessels less mature and leaky, and allow for increased extravasive/metastatic tumor potential 47, iii) increased prothrombotic events, which may be caused directly or indirectly by vessel damage as a result of therapy and allow for increased tumor cell ‘seeding’ and growth in distant organs 48, iv) altered EC adhesion molecule function, a possibility that was raised in a recent study which showed that inhibitors of αvβ3/αvβ5 administered continuously at low doses can enhance VEGF-driven angiogenesis and tumor growth 49, and v) inflammatory pathway activation, which may lead to alterations (or injury) to the endothelial microenvironment which could collectively increase both intra- and extravasive potential for tumor cells 50. Thus both tumor and host-mediated responses to antiangiogenic therapy, at least in certain instances, can facilitate proinvasive and metastatic potential after treatment in early-stage/micrometastatic disease.
While eventual enhancement of metastasis in response to an anti-cancer therapy may, at first glance, seem a counterintuitive concept (irrespective of whether mediated by tumor or host related mechanisms) it is important to note that similar findings have been reported for over 30 years with cytotoxic treatments, including radiation and various chemotherapeutic drugs 51-53. Of course a presumed difference is that chemotherapy and radiation treatments act mainly by direct tumor cytotoxicity, e.g., by non-specifically targeting proliferating cells, whereas antiangiogenic agents primarily target host processes. Furthermore, cytotoxic (and toxic) chemotherapy and radiation are administered for defined periods, e.g. 3-6 months, whereas antiangiogenic agents are (at least theoretically) meant to be administered for longer periods of time, if not indefinitely.
While many host- and tumor-mediated pathways of resistance may explain, at least in part, some of the modest benefits attained in the majority of patients treated with anti-VEGF pathway targeted agents, it remains unclear whether antiangiogenic therapy will lead to increased invasion and/or metastasis after either prolonged or short-term treatments in the clinic. To date, the literature regarding this point remains largely anecdotal and limited to case reports or small studies, but there are some clues that suggest relapsed tumors may have an altered/increased progression after therapy stops working and/or when therapy is halted altogether. For example, in many instances human GBM patients treated with bevacizumab in combination with chemotherapy experience eventual tumor relapse/regrowth accompanied by a high rate of diffuse infiltrative lesions 54-58 — a finding suggestive of an adaptive/evasive response to antiangiogenic therapy leading to increased invasiveness. As well, there are instances where discontinuation of anti-VEGF pathway targeted therapy may support preclinical observations of ‘rebound revascularization’, something which in turn could influence tumor regrowth and/or metastasis. For example, cases of tumor ‘flares’ have been reported during drug-free break periods 59 or after treatment discontinuation in RCC patients receiving either sunitinib or sorafenib 60,61, or in CRC patients treated with bevacizumab in combination with chemotherapy 62. Furthermore, in addition to primary tumor regrowth after treatment cessation with various VEGF RTKIs, increases in local foci or metastatic spread in distant organs have been noted in certain retrospective analyses 63. Importantly, there are emerging clues that some patients having failed to respond to (or been taken off) VEGF RTKI treatment, may respond again with the same drug after a break period 63 or respond when the drug is switched for another (e.g. sunitinib to sorafenib or vice versa) 64,65.
To date, one of the difficulties in uncovering (and predicting) antiangiogenic drug resistance mechanisms is a general disconnect between how such drugs are evaluated in experimental and clinical settings. For example, in most cases, patients in early phase clinical trials receiving antiangiogenic agents (or any other type of anti-cancer drug/therapy for that matter) have late-stage metastatic disease, often in multiple sites, whereas the majority of preclinical work focuses on localized primary tumors 66. Thus it is essential that future testing of antiangiogenic (and other) therapies address this gap by investigating anti-cancer agents during various stages tumor progression, especially when advanced metastatic disease is already established or, conversely, when only microscopic metastasis are present. These considerations are of particular relevance because a) metastasis is generally the main reason for patient mortality rather than primary tumor growth 67, and b) antiangiogenic agents are now being evaluated in earlier stages of disease such as in the adjuvant setting, which may involve neither primary tumors or established metastasis, but rather early-stage occult micrometastatic disease. Indeed, surprisingly few preclinical studies have tested anti-VEGF targeted pathway drugs in early (micro) and late (established) stage metastasis, and even fewer still have directly compared anti-tumor efficacy in these indications to locally grown primary tumors. In such cases, mixed results have been observed, some of which may help explain the modest benefits seen in patients. For instance, VEGF RTKIs generally have been shown to slow or stop primary tumor growth in mice but the effects on established metastatic disease range from efficacious 68 to only a marginal or negligible benefit on the overall survival of mice 69. Moreover, in micrometastatic/early stage disease, the aforementioned studies by Ebos et al and Paez-Ribes et al show that VEGF pathway targeted therapy can result in increased rates or levels of tumor invasiveness and metastasis, in certain instances. Critically, such results contrasted in both studies with the potent tumor growth inhibitory effects the same drugs and treatment schedules had on locally grown primary tumors (Ebos et al. and Paez-Ribes et al.). It is likely that various experimental conditions - such as the animal model, tumors doses, drugs, treatment duration, combinations with chemotherapy — may explain some of these differences in experimental outcomes; however it is possible that differential efficacies with antiangiogenic therapy may be observed between micro- and macro-metastatic disease. Some studies with genetically engineered mouse models of intestinal adenomas (APCmin mice) show that tumor growth can be slowed and survival prolonged after treatment with various inhibitors of the VEGF pathway 70-72. Similarly, transgenic mouse models of NSCLC, generated by mutations in Kras and conditional Lkb1 deletions, had decreased metastasis and improved survival when sunitinib treatment started 4 weeks after metastatic tumor growth induction. Yet outcome in this latter instance was not improved if treatment started 2 weeks earlier 73,74 — raising the question whether observed benefits after treatment were because of effects against established metastasis rather than microscopic disease.
Such distinctions could be important for interpretation of both preclinical and clinical trials involving antiangiogenic agents in the adjuvant setting. In limited preclinical models where primary tumors are removed and antiangiogenic drug treatment is initiated, metastatic growth could be inhibited 68,75 or accelerated 29 — depending on the tumor models and drugs used, and when treatments are initiated. What is clear is that more studies should be conducted preclinically to test anti-VEGF pathway targeted therapy in an authentic adjuvant setting, i.e., very shortly or immediately after surgical resection of a primary tumor when only microscopic minimum residual disease is present — something that might be determined by various imaging techniques and/or other measures 76. Ironically, it might be that this question will be addressed and answered first in the clinical setting. Currently there are more than 40+ adjuvant clinical trials underway involving multiple VEGF pathway inhibitors, such as sorafenib and sunitinib 77, as well as bevacizumab (typically in combination with chemotherapy), in numerous cancer types, including breast cancer, renal cancer, prostate, head and neck cancers, NSCLC, ovarian, and there are others A. With respect to bevacizumab one such trial has been completed as of 2009, e.g. a Phase III study in postoperative colorectal patients with stage II-III disease, who were treated with the anti-VEGF antibody plus chemotherapy for 1 year and 6 months, respectively. The results of this trial (NSABP-C-08; clinicaltrials.gov: NCT00096278) showed no benefit in progression free survival when assessed three years after therapy initiation 78. Interestingly, a clear benefit in favour of the bevacizumab at one year (i.e., when therapy was completed) was observed — but this benefit gradually disappeared over time. The basis for this phenomenon is unknown and clearly highlights the urgent need for undertaking preclinical studies appropriate models to study the mechanisms by which antiangiogenic treatments such as VEGF pathway targeted drugs lose their activity and/or alter tumor progression and metastasis over time.
Taken together, it is now confirmed that anti-VEGF pathway targeted therapies represent an effective treatment of cancer in certain settings, however it may be necessary to recalibrate expectations and consider improved rational strategies to overcome these limitations at all stages of tumor progression.
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